Method of producing an immunoligand/payload conjugate

ABSTRACT

The present invention relates to a method of producing an immunoligand/payload conjugate, which method encom passes conjugating a payload to animmunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof.

The present invention is related to methods of producing animmunoligand/payload conjugate

INTRODUCTION

Currently, the predominant methods to label and/or to conjugatemolecules to proteins, especially, when small-molecule payloads orlabels are concerned, involve the chemical conjugation with specificlinker molecules that covalently attach the payload to free lysineand/or cysteine amino acids of the proteins.

However, many proteins, like e.g. antibodies that are of particularinterest for immunotargeting strategies, are fairly large proteins, thatmay contain several lysine and cysteine residues. Becauselinker-mediated, chemical conjugation is a stochastic process,linker-mediated chemical ligation of payloads leads to heterogeneousmixtures of conjugated proteins that may differ in their therapeuticefficacy and/or diagnostic utility. Obviously, mixtures ofprotein-payload conjugates also represent a significant challenge in theregulatory approval process for therapeutic conjuagtes, asbatch-to-batch variation and/or variations in the active pharmaceuticalingredient (API) are negatively viewed by regulatory authorities due topotential safety concerns

In addition, if a defined ratio of payload to protein is desired, it isoften necessary to purify the conjugate with the desired conjugationstoichiometry. This is not only tedious, but can significantly add tothe cost-of-goods in the manufacturing process, as often only a fractionof the linker-mediated conjugated protein represents the desired ratioof payload conjugation. This is particularly true for therapeuticallyrelevant antibody/drug conjugates (ADCs), where depending on the toxinemployed, 3 to 4 toxin molecules appear to be advantageous, butantibodies with no toxin coupled to up to 8 toxins per antibody coupledare found in typical linker-mediated chemical conjugation reactions(Panowski et al. (2014)).

Despite of the limitations described above, all antibody/drug conjugatescurrently in clinical trials, or approved by the health authorities forthe therapy of disease, have been generated by linker-mediated chemicalconjugation of toxic small-molecule drugs to antibodies (Lambert (2012)or Mullard (2013)).

It is widely acknowledged in the industry and by scientific experts inthe field, that site-specific and stoichiometric conjugation ofmolecular payloads, including toxin or label molecules to immunoligandswould have significant advantages in comparison to chemical,linker-mediated conjugation. This is evidenced by attempts to target thechemical conjugation to specific amino acids in the protein structure(Panowski et al. (2014)).

On one hand, this is attempted by mutating certain positions in theprotein structure to delete unwanted and/or to provide desiredconjugation sites (i.e. lysine and/or cysteine residues) to which thelinker-ligation can be targeted (McDonagh et al. (2006) or Junutula etal. (2008)).

On the other hand, control of chemical conjugation to proteins isattempted by incorporation of unnatural amino acids at certainpositions, like selenocysteine, p-azidophenylalanine, oracetylphenylalanine (Hofer et al. (2009), Axup et al. (2012), or Lemke(2011)).

However, all of these approaches change the primary amino acid sequenceof the protein to be conjugated, and may result in undesired functionalproperties. Furthermore, the incorporation of unnatural amino acids, asdescribed above, is often low efficient, and does not allow for aquantitative incorporation of specific labeling sites to proteins.

SUMMARY OF THE INVENTION

Therefore, there is an urgent need in the industry to overcome the knownissues of stochastic conjugation methods in particular for thegeneration of therapeutically relevant immunoconjugates, including, butnot limited to ADCs.

It is thus one object of the present invention to provide an efficientmethod for conjugating immunoligands and payloads, e.g., drugs, toxins,cytokines, markers, or the like, preferably full-length monoclonalantibodies to small-molecular weight toxins, for the generation ofsite-specifically conjugated antibody drug conjugates (ADCs).

It is another object of the present invention to createimmunoligand/payload conjugates, which have better efficacy and/or canbe produced with higher reproducibility.

It is another object of the present invention to allow the conjugationof payloads to immunoligands in a site-specific and/or sequence specificmanner.

It is another object of the present invention to createimmunoligand/payload conjugates which preserve the characteristicfeatures of its components, e.g., target affinity, target specificity,target sensitivity, solubility, pharmacological function and the likeThese objects are achieved by the subject matter of the independentclaims, while the dependent claims as well as the specification disclosefurther preferred embodiments.

DEFINITIONS

As used herein, the term “immunoligand” is meant to define an entity, anagent or a molecule that has affinity to a given target, e.g., areceptor, a cell surface protein, a cytokine or the like. Suchimmunoligand may optionally block or dampen agonist-mediated responses,or inhibit receptor-agonist interaction. Most importantly, however, theimmonoligand may serve as a shuttle to deliver a payload to a specificsite, which is defined by the target recognized by said immunoligand.Thus, an immunoligand targeting, for instance, but not limited to areceptor, delivers its payload to a site which is characterized byabundance of said receptor. Immunoligands include, but are not limitedto, antibodies, antibody fragments, antibody-based binding proteins,antibody mimetics, receptors, soluble decoy receptors, scaffold proteinswith affinity for a given target and ligands of receptors.

“Antibodies”, also synonymously called “immunoglobulins” (Ig), aregenerally comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains, and are therefore multimeric proteins, or anequivalent Ig homologue thereof (e.g., a camelid nanobody, whichcomprises only a heavy chain, single domain antibodies (dAbs) which canbe either be derived from a heavy or light chain); including full lengthfunctional mutants, variants, or derivatives thereof (including, but notlimited to, murine, chimeric, humanized and fully human antibodies,which retain the essential epitope binding features of an Ig molecule,and including dual specific, bispecific, multispecific, and dualvariable domain immunoglobulins; Immunoglobulin molecules can be of anyclass (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2) and allotype.

An “antibody-based binding protein”, as used herein, may represent anyprotein that contains at least one antibody-derived V_(H), V_(L), orC_(H) immunoglobulin domain in the context of other non-immunoglobulin,or non-antibody derived components. Such antibody-based proteinsinclude, but are not limited to (i) F_(e)-fusion proteins of bindingproteins, including receptors or receptor components with all or partsof the immunoglobulin C_(H) domains, (ii) binding proteins, in whichV_(H) and or V_(L) domains are coupled to alternative molecularscaffolds, or (iii) molecules, in which immunoglobulin V_(H), and/orV_(L), and/or C_(H) domains are combined and/or assembled in a fashionnot normally found in naturally occuring antibodies or antibodyfragments.

An “antibody drug conjugate” (ADC), as used herein, relates to either anantibody, or an antibody fragment, or and antibody-based bindingprotein, coupled to a small molecular weight active pharmaceuticalingredient (API), including, but not limited to a toxin (including e.g.,but not limited to, tubulin inhibitors, actin binders, RNA polymeraseinhibitors, DNA-intercalating and modifying/damaging drugs), a kinaseinhibitor, or any API that interferes with a particular cellular pathwaythat is essential for the survival of a cell and/or essential for aparticular physiologic cellular pathway.

An “antibody derivative or fragment”, as used herein, relates to amolecule comprising at least one polypeptide chain derived from anantibody that is not full length, including, but not limited to (i) aFab fragment, which is a monovalent fragment consisting of the variablelight (V_(L)), variable heavy (V_(H)), constant light (C_(L)) andconstant heavy 1 (C_(H)1) domains; (ii) a F(ab′)2 fragment, which is abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a heavy chain portion of a F_(ab)(F_(d)) fragment, which consists of the V_(H) and C_(H)I domains; (iv) avariable fragment (F_(v)) fragment, which consists of the V_(L) andV_(H) domains of a single arm of an antibody, (v) a domain antibody(dAb) fragment, which comprises a single variable domain; (vi) anisolated complementarity determining region (CDR); (vii) a single chainF_(v) Fragment (scF_(v)); (viii) a diabody, which is a bivalent,bispecific antibody in which V_(H) and V_(L) domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with the complementarity domains of another chainand creating two antigen binding sites; and (ix) a linear antibody,which comprises a pair of tandem F_(v) segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementarity lightchain polypeptides, form a pair of antigen binding regions; and (x)other non-full length portions of immunoglobulin heavy and/or lightchains, or mutants, variants, or derivatives thereof, alone or in anycombination.

The term “modified antibody format”, as used herein, encompassesantibody-drug-conjugates, Polyalkylene oxide-modified scFv, Monobodies,Diabodies, Camelid Antibodies, Domain Antibodies, bi- or trispecificantibodies, IgA, or two IgG structures joined by a J chain and asecretory component, shark antibodies, new world primateframework+non-new world primate CDR, IgG4 antibodies with hinge regionremoved, IgG with two additional binding sites engineered into the CH3domains, antibodies with altered Fc region to enhance affinity for Fcgamma receptors, dimerised constructs comprising CH3+VL+VH, and thelike.

The term “antibody mimetic”, as used herein, refers to proteins notbelonging to the immunoglobulin family, and even non—proteins such asaptamers, or synthetic polymers. Some types have an antibody-likebeta-sheet structure. Potential advantages of “antibody mimetics” or“alternative scaffolds” over antibodies are better solubility, highertissue penetration, higher stability towards heat and enzymes, andcomparatively low production costs.

Some antibody mimetics can be provided in large libraries, which offerspecific binding candidates against every conceivable target. Just likewith antibodies, target specific antibody mimetics can be developed byuse of High Throughput Screening (HTS) technologies as well as withestablished display technologies, just like phage display, bacterialdisplay, yeast or mammalian display. Currently developed antibodymimetics encompass, for example, ankyrin repeat proteins (calledDARPins), C-type lectins, A-domain proteins of S. aureus, transferrins,lipocalins, 10th type III domains of fibronectin, Kunitz domain proteaseinhibitors, ubiquitin derived binders (called affilins), gammacrystallin derived binders, cysteine knots or knottins, thioredoxin Ascaffold based binders, nucleic acid aptamers, artificial antibodiesproduced by molecular imprinting of polymers, peptide libraries frombacterial genomes, SH-3 domains, stradobodies, “A domains” of membranereceptors stabilised by disulfide bonds and Ca2+, CTLA4-based compounds,Fyn SH3, and aptamers (oligonucleic acid or peptide molecules that bindto a specific target molecules)

In case the immunoligand is not a protein or a peptide, e.g., if its anaptamer, it should preferably be provided with a peptide tag in order toprovide a suitable substrate for the enzymatic conjugation disclosedfurther herein.

“Conjugation”, as used herein, relates to the covalent association of amolecule to another molecule by formation of a covalent bond.

An “immunotoxin”, as used herein, relates to an immunoligand conjugatedto a protein or polypeptide representing a toxin, including, but notlimited to bacterial toxins, e.g. diphteria-toxin A, Pseudomonasexotoxin, botulinum toxin, or e.g. proteinaceous venoms frominvertebrates (e.g. but not limited spiders, scorpions, molluscs,jelly-fish), or vetrebrates (e.g., but not limited to snakes), orfunctional fragments thereof.

The term “low molecular-weight payload” as used herein, represents apayload with a molecular weight not exceeding 2'′500 Dalton.

The term “payload”, as used herein, represents any naturally occuring orsynthetically generated molecule, including small-molecular weightmolecules or chemical entities that can chemically be synthesized, andlarger molecules or biological entities that need to be produced byfermentation of host cells and that confer a novel functionality to animmunoligand specific for binding to targets or antigens.

The term “small molecular weight toxin”, as used herein, means acytotoxic compound of small molecular weight not exceeding a molecularweight of 2′500 Dalton that is cytotoxic to mammalian cells.

A “transpeptidase”, as used herein, is an enzyme or a catalytic domainof an enzyme or a protein that is able to catalyze the breakage ofpeptide bonds and subsequently either directly, or by way of severalreaction intermediates, the formation of novel peptide bonds, such thatthe energy of the first peptide bond is preserved during the reactionand transfered to a new peptide bond. Preferably, said transpeptidasespreferably connect the C-terminus of one peptide or protein with theN-terminus of another peptide or protein. Due to the formation of a newpeptide bond, these enzymes or functional domains are also refered to as“protein ligases”, “peptide ligases”, or nicknamed “protein or peptidestaplers”. Such protein ligases comprise, but are not limited to sortaseenzymes, inteins and split-inteins.

As used herein, the term “sequence-specific transpepeptidase” is meantto define a transpeptidase which needs at least one substrate peptide orprotein with a given peptide sequence as recognistion sequence(N-terminally and/or C-terminally) to connect said substrate peptide orprotein to another peptide or protein, or a small-molecular weightcompound containing a peptide or protein component

As used herein, the term “site-specific transpepeptidase” is meant todefine a transpeptidase which has a specific site in at least onesubstrate peptide or protein which it uses to conjugate to anotherpeptide or protein, or a small-molecular weight compound containing apeptide or protein component.

BACKGROUND AND GENERAL DESCRIPTION OF THE INVENTION

The invention discloses methods that utilize site-specifictranspeptidases, e.g., sortase enzymes and split-inteins, tosite-specifically and selectively conjugate payloads, preferably smallmolecular weight toxins to immunoligands, preferably antibodies, for thegeneration of immunoligand payloads, preferably antibody drug conjugates(ADCs). The preferred payloads are small molecular weight toxinsmodified with short, preferably less than 13 (thirteen) amino acid longsynthetic amino acid sequence, which renders them as substrates forsortase enzymes or split intein mediated covalent conjugation either atthe N- or C-terminus of the immunoligands (FIGS. 1 & 3). Thisconjugation is achieved in a site-specific manner and with definedstoichiometry, which is a distinguishing feature to conventionalchemical conjugation of payloads to immunoligands, where the conjugationis a stochastic process, as disclosed further above.

The invention further discloses site specific transpeptidase, e.g.sortase or split-intein mediated conjugation of multimericimmunoligands, preferably antibodies specifically with two differenttoxin molecules or other labels using different modifications of thesubunits of the multimeric protein, e.g. antibody heavy and lightchains, and different payloads modified with different, short amino acidstretches specific for different transpeptidases, in order to conjugateat least two different functional payloads to the multimericimmunoligand (FIG. 6).

The invention further discloses methods to add affinity purificationand/or detection tags to the N- or C-termini of the immunoligands, whichundergo enzyme-mediated transpeptidation. such that the removal of theaffinity purification and/or detection tag can be utilized to select forimmunologands with complete (100%) conjugation of the payload to themodified binding protein, by means of affinity resins that retainimmunoligands that have not been completely conjugated, and thereforestill retain the additional affinity purification and/or detection tag(FIG. 4).

The invention further discloses immunoligands in which a catalytictranspeptidase domain is directly fused to the N- or C-terminus of theprotein to be conjugated, such that the transpeptidation activity isintegral part of immunoligand to be conjugated, and no additionalsoluble sortase enzyme needs to be provided in the course of thetranspeptidase-mediated conjugation reaction (FIG. 5).

All of these embodiments mentioned above allow the site-specific andstoichiometrically controlled conjugation of any payload, includingsmall molecule toxins (chemical entities), toxic proteins, orfluorescent labels, preferably small molecular weight toxins toimmunoligands, including preferably antibodies, which is superior tostandard chemical conjugation of payloads to proteins by chemical linkerchemistry methods, which cannot be controlled for conjugation ratio andsite. Therefore, for the generation of antibody drug conjugates (ADCs)conjugation of toxic payloads by transpeptidases, preferably sortaseenzymes and split inteins to antibodies will lead to more homogeneousproducts with expected improved therapeutic properties for cancertherapy (FIG. 12).

The enzymatic conjugation of payloads to immunoligands by sortaseenzymes and split-intein allows site-specific and stoichiometric payloadconjugation to proteins and immunoligands, lowering cost-of-goods andproviding homogeneous immunoligand-payload conjugates, especially as theselectivity of the transpeptidases allows the conjugation of payloads toimmunoligands in crude cell culture supernatant, and does not requirepurified components as in traditional linker-mediated chemicalconjugation. Therefore, the use of sequence-specific transpeptidases forconjugation of payloads to immunoligands could significantly lower thecost of goods in immunoligand-payload, and particularly ADCmanufacturing.

The first type of transpeptidase disclosed herein, the sortase enzymes,has been identified in a variety of gram-positive bacteria, likeStaphylococcus, Streptococcus and Pneumococcus species, and catalyse thecoupling of virulence factors to cell wall proteoglycans, in order tochange the surface signature of the bacteria for evading an efficientimmune response by the infected host (Mazmanian et al. (1999)). SortaseA enzyme of the gram-positive bacterium Staphylococcus aureus has beencharacterized first (Ton-That et al. (1999)) and has subsequently beencharacterized further as a tool for many protein modifications (Tsukiji(2009)). The attraction of sortase enzymes is that the two molecules tobe conjugated only require to be modified or expressed on one hand witha short 5 amino-acid long peptide tag (sortase tag, LPXTG in case ofStaphylococcus aureus sortase A, X being any of the 20 naturallyoccuring aminoacids), and a short, preferably 3 to 5 amino acid longglycine stretch (Antos et al. (2009a)) (FIG. 1), which can easily beadded to each of the molecules to achieve either N-terminal orC-terminal conjugation of proteins. This allows to utilize the system onone hand for the coupling or conjugation of two proteins, but also forthe conjugation of smaller molecules to proteins.

The second type of transpeptidase resulting in peptide-bond cleavage andformation, is represented by the so-called inteins, which haveoriginally been discovered as protein introns, that can remove (splice)themselves out of precursor proteins by cleavage of peptide bonds andformation of new peptide-bonds (Xu et al. (1993)) (FIG. 2a ). Inteinscan also occur separated into N-intein and C-intein domains (so-calledsplit-inteins) and attached to independent proteins that cansubsequently catalyze the trans-splicing of the extein domains (FIG. 2b). Split-inteins have been utilized for the covalent coupling ofN-extein and C-extein moieties, and also the purification and/orcircularization of proteins (Elleuche (2010)). However, in order toutilize split-inteins also for the conjugation of small moleculepayloads, it is necessary to utilize split inteins that function ifeither the N-intein or the C-intein domain can be redused to few aminoacids, that can easily be added to molecules of any size by chemicalsynthesis, similar to the short at preferably 3 glycine stretch requiredfor sortase-mediated transpeptidation. With the development of theartificial Ssp GyrB S11 split-intein, in which the C-intein domain onlycomprises six amino acids (Sun et al. (2004)), this condition has beenmet and this split-intein has been utilized for the C-terminal labelingof proteins with biotin (Volkmann et al. (2009)) (FIG. 3a ). Likewise,the development of a short 11 amino acid long N-intein from Ssp DnaXsplit-intein allows the N-terminal conjugation of proteins with anymolecule, if such 11 amino acid long stretch is added by chemicalsynthesis to a payload of choice (FIG. 3b ).

Therefore, one aspect of the invention is either to add a short,preferably 3 to 5 glycine amino-acid glycine stretch, or a short 12amino-acid GVFVHNSXXXXX amino acid stretch (X any naturally occuring orartificial amino acids), containing a 6 amino acid C-int domain of SspGyrB or a 11 amino-acid N-int domain of Ssp DnaX to a payload-molecule,which is sufficient to allow the respective trasnpeptidase to conjugatethe modified payload to proteins and immunoligands, preferablyantibodies that, respectively, contain a sortase enzyme recognitionmotif, e.g. LPXTG in case of utilization of Staphylococcus aureusSortase A, or a 150aa N-int domain in case of utilization of Ssp GyrBsplit intein, or a 139 aa C-int domain in case of utilization of SspDnaX split intein (see FIGS. 1 & 3).

The addition of short stretches of amino acids, like e.g. 3 or 5 glycineresidues to a small molecular weight toxins as required for sortasemediated conjugation, or 12 amino acids as required for split-inteinmediated conjugation, has been found to add to the water-solubility ofcertain hydrophobic toxin molecules (data not shown), such that theamino acid-toxin adduct can be dissolved in the physiologic buffer,ensuring optimal sortase or split-intein conjugation. This preventsstress on the strutural integrity of large protein molecules,particularly antibodies that can easily be denatured by exposure toorganic solvents and non-physiologic pH often associated withtraditional linker chemistry and conjugation. In addition, conjugationof hydrophobic toxin molecules to large proteins, particular antibodiescan induce certain levels of protein aggregation. Also this may beimproved by using transpeptidases, particularly sortase enzymes, becausefurther hydrophilic amino acids remain in the enzymatically generatedconjugate, reducing the propensity for aggregation of large protein, orantibody drug conjugates.

Sortase enzymes have been widely described in the prior art forprotein-protein or protein-peptide ligations (Mao et al. (2004),Parthasarathy et al. (2007) or WO2011/133704A2), even includingcircularization of proteins (Antos et al. (2009b)). The applications ofsortase protein or peptide ligation also included protein or peptideligation using antibody fragments, like Fab- and scFv-fragments withprotein- or peptide labels (Möhlmann et al. (2011), Madej et al. (2012),or US2010/0055761A1 and WO2012/142659A1). Even two prior art documentswere published, in which full-length antibodies have beensortase-ligated to proteins (Levary et al. (2011), e.g. EGFP, albumin,gelonin were conjugated to the light chain of an antibody), or in whichfull-length antibodies have been sortase-ligated to short peptides (Sweeet al. (2013)). However, no prior art document could be identifieddemonstrating the sortase-mediated conjugation of small-molecular weighttoxins, like e.g. auristatins or maytansins and the like, to full-lengthantibodies or antibody fragments. In particular no prior art documentscould be identified, in which generation of ADCs with small molecularweight toxins has been been disclosed resulting in ADCs with smallmolecular weight toxins homogeneously conjugated to either IgH or IgLchains (drug-to-antibody ratio 2), or to IgH and IgL chains (drug toantibody ratio 4), as disclosed herein.

While the prior art also discloses the modification of non-proteinsubstrates with glycine residues such that they could be used forsortase modification of simple, single-subunit proteins or peptides(Tsukiji (2009), or WO2007/108013A3, respectively), the more challenginghomogeneous conjugation of non-protein substrates, preferably smallmolecular weight toxins, to multimeric proteins, preferrably antibodies,has not been described before, despite the fact that sortase enzymemediated protein or peptide ligation has been in the prior art for manyyears.

Moreover, the conjugation of multimeric proteins, particularlyfull-length monoclonal antibodies with two different payloads,preferrably two different small molecular weight toxins as disclosedherein, has not been described in the prior art before, despite the factthat sortase enzyme mediated protein or peptide ligation has been in theprior art for many years (Panowski et al. (2014)).

It is known from the prior art that sortase enzymes may acceptsubstrates that contain a minimum of 3 glycine amino acids(Parthasarathy et al. (2007), therefore the invention may includepayloads that contain at least three (3) glycine amino acid residuesadded to the payload molecule of interest, although even one or twoglycine residues may be sufficient, and should be comprised by themethod disclosed herein. In case of small molecular weight payloads theaddition of few glycine amino acid residues can be achieved byconventional synthetic peptide chemistry, as described herein. In caseof proteins glycine residues can be added either by adding codons for anumber of glycine residues, preferably at least three glycine residues,in-frame to the open reading frame of the protein, or by conventionalsynthetic peptide chemistry such that the recombinant protein containsat least three N-terminal glycine amino acid residues.

It is known from the literature that different Sortase enzymes, e.g.Sortase B from Staphylococcus aureus, or Sortases from othergram-positive bacteria recognize different pentapeptide motifs, whichdiffer from the LPXTG sortase A recognition motif (X=any amino acid)from Staphylococcus aureus (Spirig et al. (2011)). Therefore, theinvention shall also include the concept of adding other sortaserecognition motifs to proteins and immunoligands, including preferablyantibodies, that differ from the Staphylococcus aureus sortase Arecognition motif LPXTG, in order to prepare them for sortaseconjugation with different cognate sortase enzyme of differentgram-positive bacterial species. Therefore, proteins and immunoligands,preferably antibodies, can also be expressed with a different sortaserecognition motif, e.g. a NPQTN pentapeptide motif specific for sortaseB from Staphylococcus aureus which can then be conjugated to glycinemodified payloads.

In an another aspect of the invention, multimeric immunoligands,preferably but not limited to antibodies, which are composed ofimmunoglobulin heavy and light chains, allow the utilization of saiddifferent sortase recognition sequences added to the differentpolypeptides of such multimeric proteins (in case of antibodies addingdifferent sortase recognition sequences to the antibody heavy and lightchains), in order to allow conjugation of different payloads to saiddifferent polypeptides by performing sequential conjugations withGly_(n)-tagged payloads (n>2) in the presence of the respective sortaseenzyme (FIG. 6b ). For this, an antibody needs to be expressed withdifferent C-terminal modifications at heavy and light chains comprisingdifferent sortase recognition motifs for different sortase enzymes. Suchan antibody can then sequentially be conjugated to two differentpayloads containing a glycine modification as described further above.

This format may have the advantage that ADCs specifically be loaded withtwo different toxins, preferably interfering with a different cellularpathway will be more potent in cancer cell killing, because it is moredifficult for a targeted cancer cell to evade the attack of two toxinscomprised in the ADCs.

It is clear to a person skilled in the art, that a sortase pentapeptiderecognition motif, like the Staphylococcus aureus sortase A LPXTG motif,can be added selectively to individual polypeptides of multimericimmunoligands, in order to provide desired conjugation sites. Forinstance, in the case of antibodies, this allows the generation ofmodified antibodies, either only containing sortase recognition motifsadded to the heavy chains (resulting in two payloads per antibodyconjugation), or only containing sortase recognition motifs added to thelight chains (resulting in two payloads per antibody conjugation), orcontaining sortase recognition motifs added to the heavy and the lightchains (resulting in four payloads per antibody conjugation). Thesedesigned variations will allow specific conjugation of payloads toantibodies by sortase enzymes either to the heavy chains alone(generating ADCs with drug to antibody ratio of 2, i.e. DAR2), or to thelight chains alone (generating ADCs with drug to antibody ratio of 2,i.e. DAR2), or simultaneously to the heavy and the light chains(generating ADCs with drug to antibody ratio of 4, i.e. DAR4). This way,the conjugation sites and stoichiometries for antibodies can be variedin a controlled fashion, either generating two payload conjugations perantibody heavy or light chain, or generating four payload conjugationsper antibody by addition of the payload to the heavy and the lightchains.

Similar to the above-described variations in conjugation sites andstoichiometries using different sortase recognition motifs and sortaseenzymes in multimeric proteins or immunoligands, it is a further aspectof the invention to conjugate different payloads to different polpeptidechains of multimeric proteins combining sortase-mediated andsplit-intein mediated conjugation. This concept allows the simultaneousconjugation of different payloads to different polypeptide chains ofmultimeric proteins and immunoconjugates in one step, because differenttranspeptidases and substrates are being employed (FIG. 6a ).

It is to be understood that the above-mentioned conjugation of twodifferent payloads to a multimeric protein, preferably an antibody,which is composed of each two disulphide linked heavy and lights chains,can either be accomplished by combining sortase enzyme mediatedconjugation with split intein mediated conjugation, as depicted, in FIG.6a , but that it is also possible to conjugate two different payloads toa multimeric protein, preferably an antibody, by utilizing two differentsortase enzymes, recognizing different sortase peptide motifs, forinstance sortase A and sortase B from Staphylococcus aureus, asmentioned further above (FIG. 6b ). However, this may also includesortase enzymes of other sortase classes (e.g. sortases C, D, E, F), orsortase enzymes from other bacterial species, differing in their sortasemotif specificity.

Sortase-mediated conjugation of payloads to proteins and immunoligandscan be achieved either by providing sortase recognition motif taggedproteins and at least tri-glycine tagged payloads and addingenzymatically active sortase enzyme or a functional fragment thereof asa soluble enzmye. In another aspect of the invention the enzymaticallyactive domain of sortase enzyme can also be provided as a domain fusedto either the N- or C-terminus of the protein. In this variation, is isadvantageous, but not mandatory, to add the sortase enzymatic domaineither N-terminal to an N-terminal sortase recognition motif, orC-terminal to a C-terminal sortase recognition motif. Both possibilitiesensure that the after the reaction with a glycine-tagged payload, thatthe enzymatic sortase domain is removed from the protein in the courseof the reaction (FIG. 5).

This variation of applying sortase-mediated conjugation of payloads toproteins is similar in concept to split-intein mediated conjugation ofpayloads, where the enzymatically active N-intein domains of splitinteins are tethered to the protein to be conjugated, in order to definethe conjugation site in the protein.

Similar to the large number of different sortase transpeptidases withdifferent substrate specificity that have been identified in theliterature (Spirig et al. (2011)), there is also a large and growingnumber of split-inteins known from different species and proteins withdifferent N-intein and C-intein sequences required for transpeptidationthat can be retrieved from the so-called InBase database (Perler (2002).Therefore, while the examples of split-intein mediated conjugation ofimmunoligands with payloads disclose the prefered Ssp GyrB S11 splitintein (Volkmann et al. (2009)), because the C-intein domain can bereduced to a short, linear 6-mer amino acid stretch, split-inteinmediated conjugation of payloads to proteins and immunoligands can alsobe achieved with other split inteins from the InBase database, as longas the N-intein or C-intein domains are short enough (preferably shorterthan 13 amino acids) to easily allow peptide synthesis and addition toany payload molecule of choice. However, it is clear to a person skilledin the art that in the case of protein payloads, C-intein domains of anysize may be fused to the protein payload by genetic fusion to the ORF ofthe protein payload of interest, and there is no mechanistic advantageof using split-inteins with small (<13 amino acids) N-intein or C-inteindomains.

However, if synthetic small-molecule payloads are to be conjugated toproteins and immunoligands, then a small N-int or C-int domain of lessthan 13 amino acids as disclosed herin are advantageous, as in the caseof the prefered C-int of the Ssp GyrB S11 split intein, of the N-int ofSsp DnaX, because such a short peptide can synthetically be added to anysynthetic small molecule weight payload by standard synthetic chemistry.

Sortase-mediated and split-intein mediated conjugation of payloads canbe performed at either the N- or the C-termini of proteins andimmunoligands. This is only dependent on how the sortase-motif/glycinestretch and N-intein/C-intein domains are positioned at protein andpayload (FIG. 1).

In the case of antibodies, which are the preferred immunoligands, it ispreferred to conjugate the payloads to the C-termini of the antibodies,because this positions the payloads most distally to the antigen-bindingsites of the antibody. However, this preference shall not be interpretedby way of limitation, and it may be advantageous to conjugate payloadsto the N-terminus of other immunoligand molecules, like e.g. antibodymimetics, in which the functional binding domains are not located at theN-terminus of the molecule.

Another aspect of the invention is to improve the efficiency of sortaseand split-intein conjugation of payloads to proteins and immunoligandsby adding affinity purification or detection tags, like e.g., but notlimited to small peptide tags (e.g. histidine tags, strep-tag, MYC-tagor HA-tag) or larger protein affinity purification tags (e.g.maltose-binding protein (MBP) tag, Glutathione-S-transerase (GST) tag,or Chitin-binding tag) distal to the sortase recognition motif or thesplit-intein domain fused to the protein or immunoligand of interest.With this aspect of the invention the affinity purification tag will beremoved from the immunoligand to be conjugated as part of thetranspeptidation reaction. This can be exploited to enrich fully payloadconjugated immunoligands, as unreacted proteins and immunoligands, thatstill contain the affinity purification tag, can be removed by bindingto a suitable affinity resin, while completely payload conjugatedproteins and immunoligands will no longer contain the affinitypurification tag, and can thus be specifically separated from theunreacted immunoligand substrates. This aspect of the invention isparticularly powerful in the context of multimeric proteins andimmunoligands, like the preferred antibodies, in which several payloadsneed to be conjugated. The use of affinity purification tags locateddistal to the sortase or intein transpeptidase conjugation site ensuresthat one can remove proteins and immunoligands in which the affinitypurification tag is still present due to incomplete payload conjugation(FIG. 5).

In comparison to chemical conjugation, this provides a significantadvantage in the process to obtain homogeneous immunoligand/payloadconjugates, and preferably ADCs in which small molecular weight toxinsare site specifically conjugated to the C-termini of antibody heavyand/or light chains.

Generally, the disclosed method provides a novel and efficient method tosite-specifically and stoichiometrically conjugate payloads, preferablysmall molecular weight toxins to immunoligands, preferably antibodies,by which defined immunoligand/payload conjugates, preferably ADCs aregenerated, that are useful for the therapy of diseases, preferably ofcancer. The method may also be utilized for the generation ofimmunoligand/payload conjugates useful for the diagnosis of diseases,preferably oncology diseases. The novel method allows generationcovalent immunoligand/payload conjugates by utilization of peptide-bondbreaking and forming enzymes (transpeptidases), including sortaseenzymes and split-inteins, or catalytically active fragments thereof.Said enzymes can catalyze the covalent and site-specific conjugation ofpayloads containing short amino acid stretches (preferably shorter than13 amino acids) either to the N- or C-termini of immunoligands which aresuitably modified allowing sortase and split-inteins to break and toform peptide bonds in the course of the reaction. Immunoligands arepreferably antibodies, for the site-specific conjugation of smallmolecular weight toxins, in order to generate antibody drug conjugates(ADCs) with defined antibody payload, or drug to antibody ratios.

EMBODIMENTS OF THE INVENTION

According to the invention, a method of producing animmunoligand/payload conjugate is disclosed, which method encompassesconjugating a payload to an immunoligand by means of a sequence-specifictranspeptidase, or a catalytic domain thereof.

According to a preferred embodiment of the invention, the payload and/orthe immunoligand either

a) consists, entirely, of a protein or peptide

b) comprises at least one protein or peptide domain, or

c) comprises at least one peptide chain

and, further, the protein or peptide or domain comprises, preferably, anamino acid sequence that can be detected by the sequence-specifictranspeptidase, or a catalytic domain thereof.

This means, for example, that, in case the payload and/or theimmunoligand is a protein, it means that said protein comprises, at itsN- or C-terminus, an amino acid sequence which can be detected by thesequence-specific transpeptidase. If such amino acid sequence is lackingto the naïve protein, it can be fused to the N- or C-terminus of saidprotein by recombinant methods known in the art.

In case the payload and/or the immunoligand is not a protein, such aminoacid sequence which can be detected by the sequence-specifictranspeptidase, is to be conjugated to the former by conventionalchemical crosslinking methods known in the art.

Additional functionalities may be incorprated between the recognitionsequence for a specific transpeptidase and the payload. This can berealized by chemical structures either being categorized by beingcleavable (e.g. containing hydrazone, or disulfide chemistry, orspecific peptide sequences for intracellular proteases) or beingnon-cleavable (e.g. containing thioether chemistry) followinginternalization into cells.

Chemical structures containing hydrazone chemistry can selectively becleaved within the intracellular compartment of lysosomes (lower pHcompared to the systemic blood circulation).

Peptide linkers have the potential to be selectively cleaved bylysosomal proteases (e.g. cathepsin-B) and have demonstrated increasedserum stability and improved anti-tumor effects compared to hydrazonelinkers. Valine-citruline (Val-Cit) pairs are the most commonly usedpeptide linkers and are ideally suited to work with the auristatinfamily of drugs such as monomethyl auristatin E (MMAE).

Non-cleavable Linkers have long been overlooked as researchers wereconvinced the cleaving of the linker was the most reasonable way to freethe drug. However, conjugates can, upon binding to a membrane receptor,get rapidly internalized and once internalized, the immunoligand can bedegraded to the point where the payload, e.g., the drug is exposed. Asone prominent example, thioether linkers, use the SMCC(N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate) linker(FIG..

All of theses appaoraches have in common that there is no truesite-specificty of the coupling reaction. Because linker-mediated,chemical conjugation is a stochastic process, linker-mediated chemicalligation of payloads leads to heterogeneous mixtures of conjugatedproteins that may differ in their therapeutic efficacy and/or diagnosticpotential. Obviously, mixtures of protein-payload conjugates alsorepresent a significant challenge in the regulatory approval process fortherapeutic conjuagtes, as batch-to-batch variation and/or variations inthe active pharmaceutical ingredient (API) are negatively viewed byregulatory authorities due to potential safety concerns.

Non-cleavable Linkers have long been overlooked as researchers wereconvinced the cleaving of the linker was the most reasonable way to freethe drug. However, conjugates can, upon binding to a membarne receptor,get rapidly internalized and once internalized, the immunoligand can bedegraded to the point where the payload, e.g., the drug is exposed. Oneprominent example, thioether linkers, use the SMCC(N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate) linker(See FIG. 14a , structure 2).

All of theses approaches have in common that there is no truesite-specificty of the coupling reaction. Because linker-mediated,chemical conjugation is a stochastic process, linker-mediated chemicalligation of payloads leads to heterogeneous mixtures of conjugatedproteins that may differ in their therapeutic efficacy and/or diagnosticpotential. Obviously, mixtures of protein-payload conjugates alsorepresent a significant challenge in the regulatory approval process fortherapeutic conjuagtes, as batch-to-batch variation and/or variations inthe active pharmaceutical ingredient (API) are negatively viewed byregulatory authorities due to potential safety concerns.

According to another preferred embodiment of the invention, theimmunoligand comprised in the immunoligand/payload conjugate is at leastone selected from the group consisting of

-   -   an antibody, modified antibody format, antibody derivative or        fragment, and/or    -   an antibody mimetic

Preferably, in this embodiment, a small molecular payload is rendered assubstrate for the sequence-specific transpeptidase by coupling of apeptide of less than 13 amino acids to the small molecular payload, suchthat it can be conjugated by a transpeptidase to the C-termini of amonoclonal antibody containing C-terminal modifications recognized bysaid transpeptidases. Such C-terminal modifications may be contained oneither both heavy chains, or both light chains, or of heavy and lightchains of a full-length antibody, thereby allowing generation of asite-specifically conjugated ADC with either drug-to-antibody ratio of 2or 4 (DAR2 or DAR4).

According to another preferred embodiment of the invention, theimmunoligand binds at least one entity selected from the groupconsisting of

-   -   a receptor    -   an antigen,    -   a growth factor    -   a cytokine, and/or    -   a hormone

As used herein, the term “receptor” means a cell surface molecule,preferably a cell surface molecule that (i) binds specific, or groups ofspecific, signalling molecules (i.e. a receptor, like, e.g., the VEGFreceptor), and/or (ii) has no known ligand (i.e. an orphan receptor,like, e.g. HER2/neu). The natural receptors are expressed on the surfaceof a population of cells, or they merely represent the extracellulardomain of such a molecule (whether such a form exists naturally or not),or a soluble molecule performing natural binding function in the plasma,or within a cell or organ. Preferably, such receptor is a member of asignalling cascade that is involved in a particular pathogenic process(e.g., a receptor that belongs to a signalling cascade of a growthfactor), or is expressed on the surface of a cell or particle that isinvolved in a pathological process, e.g., a cancer cell.

As used herein, the term “antigen” means a substance that has theability to induce a specific immune response, and may include surfaceproteins or protein complexes (e.g. ion channels). Often times, antigensare associated to pathogenic entities, e.g., a cancer cell.

As used herein, the term “cytokine” refers to small cell-signalingprotein molecules that are secreted by numerous cells and are a categoryof signaling molecules used extensively in intercellular communication.Cytokines can be classified as proteins, peptides, or glycoproteins; theterm “cytokine” encompasses a large and diverse family of regulatorsproduced throughout the body by cells of diverse embryological origin.

As used herein, the term “growth factor” relates to naturally occurringsubstances capable of stimulating cellular growth, proliferation andcellular differentiation. Usually a growth factor is a protein or asteroid hormone. Growth factors are important for regulating a varietyof cellular processes.

As used herein, the term “hormone” relates to a chemical released by acell, a gland, or an organ in one part of the body that sends outmessages that affect cells in other parts of the organism. The termencompasses peptide hormones, lipid and phospholipid-derived hormonesincluding steroid hormones, and monoamines.

In case the immunoligand binds a receptor or an antigen, theimmunoligand-payload conjugate can for example be directed to a specificsite, e.g., to a pathogenic entity, e.g., a cancer cell, where thepayload, e.g. a toxin or a chemotherapeutic agent, is delivered. Thus,the systemic toxicity of the toxin or the chemotherapeutic agent isreduced, while the local concentration of the latter at the site ofaction is increased, thus provding a better efficacy while side effectsare reduced. Furthermore, a respective signalling cascade can beinhibited by the binding of the immunoligand. In case the payload is amarker the latter can thus be used to mark a specific site, e.g., acancer cell charcterized by a given surface antigen detected by theimmunoligand, for diagnosis.

In case the immunoligand binds a growth factor, a cytokine, and/or ahormone, the immunologand/payload conjugate can for example be directedto the site the growth factor cytokine or hormone usuall binds to, inoder to deliver the payload in a site-specific manner. Further, arespective signalling cascade can be inhibited by the binding of theimmunoligand.

As used herein, the term “to bind” means the well-understood interactionor other nonrandom association between immunoligands, e.g., antibodies,or antibody fragments, and their targets. Preferably, such bindingreaction is characterized by high specifity and/or sensitivity to thetarget. Preferably, the binding reaction is characterized by adissociation constant (Kd) ≤10⁻³ M, preferably ≤10⁻⁴ M, ≤10⁻⁵ M, ≤10⁻⁶M, ≤10⁻⁷ M, ≤10⁻⁸ M, ≤10⁻⁹ M, and most preferred ≤10⁻¹⁰.

Acccording to a preferred embodiment of the invention, it is providedthat at least one catalytic domain of the sequence-specifictranspeptidase is fused to the N-terminus or the C-terminus of eitherthe immunoligand or the payload.

Such fusion may take place by recombinant engineering, or by chemicalcoupling. In this embodiment, the enzymatic activity leading to thesite-specific conmjugation of the immunoligand to the payload does notneed to be added to the reaction as a separate recombinant enzyme, butis rather part of protein substrate to be conjugated.

Preferably, the sequence-specific transpeptidase is at least oneselected from the group consisting of

-   -   a sortase, or one or more fragments or derivatives thereof.    -   a spilt-intein, or one or more fragments or derivatives thereof.

In a preferred embodiment, where the transpeptidase is a sortase, thepayload, e.g., a toxin, is preferably rendered as substrate for sortaseconjugation by addition of a small number of glycine amino acidresidues, preferably 3 or 5 glycine residues.

In another preferred embodiment, where the transpeptidase is a splitintein, e.g., a Ssp GyrB split intein, the payload, e.g., a toxin isrendered as substrate for split intein conjugation by addition of lessthan 13 amino acid residues of the sequence GVFVHN-SX_(n), X being anyamino acid and n being an integer between ≥0 and ≤5.

The use of transpeptidases, preferably sortase enzymes and split inteinsfor the generation of antibody drug conjugates, in which small molecularweight toxins are conjugated to full-length antibodies, has not yet beendescribed in the prior art (Panowski et al. (2014)).

Sortase enzymes have been identified in a variety of gram-positivebacteria, like Staphylococcus, Streptococcus and Pneumococcus species,and catalyze, in vivo, the coupling of virulence factors to cell wallproteoglycans, in order to change the surface signature of the bacteriafor evading an efficient immune response by the infected host (Mazmanianet al. (1999)).

The sortase A enzyme of the gram-positive bacterium Staphylococcusaureus has been characterized first (Ton-That et al. (1999)) and hassubsequently been characterized further as a tool for many proteinmodifications (Tsukiji (2009)).

One beneficial feature of sortase enzymes is that the two molecules tobe conjugated only require short peptide tags (“sortase tags”), which incase of Staphylococcus aureus sortase A is for example LPXTG at theC-terminus of one molecule (e.g., the payload), and a short 3 to 5 aminoacid glycine stretch at the N-terminus of the other molecule (e.g., theimmunoligand, see FIG. 1). These peptide tags can either be fused to themolecules, or conjugated thereto by means of conventional crosslinkingchemistry. This allows to utilize the system on one hand for theligation of two proteins, but also for the conjugation of smallmolecular weight compounds, preferably small molecular weight toxins toproteins. In case of Staphylococcus aureus sortase B, the respectivesortase motif is NPQTN.

Inteins, which have originally been discovered as protein introns thatcan remove (splice) themselves out of precursor proteins by cleavage ofpeptide bonds and new peptide-bond formation (Xu et al. (1993)) (FIG. 2a).

Naturally occuring and artificial split-inteins involve that the inteincoding region has been split into N-intein and C-intein domains, whichcan be attached to different proteins or peptides in such way that,subsequently the trans-splicing of the extein domains (FIG. 2b ) leadsto the conjugation of the two proteins

Split-inteins have thus been utilized for the covalent coupling ofN-extein and C-extein moieties, and also for the purification and/orcircularization of proteins (Elleuche (2010)). One embodiment disclosedherein is to utilize split-inteins for the conjugation of smallmolecular weight compounds, preferably small molecular weight toxins andother small molecule labels, in which a short C-extein peptide sequenceof smaller than 13 amino acids is coupled to molecules of any size,similar to the short glycine amino acid stretch required forsortase-mediated transpeptidation.

In case of sortase enzymes addition of a short glycine stretch (>2glycine residues) to a molecule of choice is sufficient to allow themolecule to be conjugated to immunoligands containing a penta-peptidesortase recognition motif, like e.g. LPXTG in case of sortase A of S.aureus. In case of split-inteins, minimally a short 12 amino-acidGVFVHNSAGSGK amino acid stretch containing a short, 6 amino acidC-intein (GVFVHN) from Ssp GyrB and a short C-extein (here: SAGSGK) aresufficient to modify any payload molecule, preferably a small molecularweight toxin, for split-intein mediated conjugation to immunoligandscontaining the N-intein domain of the Ssp GyrB split intein (Volkmann etal. (2009)). Other split inteins, in which functional intein domains canbe reduced to small <13 amino acid long peptide stretches may beutilized as well.

Even if, in the literature, split-enzymes are not always referred to asenzymes, they qualify as such, because the reaction they catalyzeresults in the breakage of a peptide bond and the formation of a newpeptide bond and this can be viewed as transpeptidases, because theenergy of an existing peptide bond is transfered to a new peptide bond.

Other than chemical conjugation, the transpeptidase-mediated conjugationoccurs under physiologic aqueous buffer conditions and physiologictemperatures, thereby minimally affecting the protein or antibodyintegrity in the conjugation reaction. This feature ensures optimalfunctionality of the resulting conjugate

Acccording to another preferred embodiment of the invention, it isprovided that the payload comprised in the immunoligand/payloadconjugate is at least one selected from the group consisting of

-   -   a marker    -   a processing tag, and/or    -   a drug.

The term “marker” (also called “detection tag”), as used herein, mayrefer to any molecule or moiety that comprises one or more appropriatechemical substances or enzymes, which directly or indirectly generate adetectable compound or signal in a chemical, physical or enzymaticreaction.

The term “processing tag” as used herein, may encompass affinity tags,solubilization tags, chromatography tags and epitope tags. Affinity tags(also used as purification tags) are appended to proteins so that theyallow purifification of the tagged molecule from their crude biologicalsource using an affinity technique. These include chitin-binding protein(CBP), maltose binding protein (MBP), and glutathione-S-transferase(GST). The poly(His) tag, preferably a 6×His tag, is a widely-usedprocessing tag; it binds to metal matrices. Solubilization tags areused, especially for recombinant proteins expressed inchaperone-deficient species such as E. coli, to assist in the properfolding in proteins and keep them from precipitating. These includethioredoxin (TRX) and poly(NANP). Some affinity tags have a dual role asa solubilization agent, such as MBP, and GST.

Chromatography tags are used to alter chromatographic properties of theprotein to afford different resolution across a particular separationtechnique. Often, these consist of polyanionic amino acids, such asFLAG-tag.

Epitope tags are short peptide sequences which are chosen becausehigh-affinity antibodies can be reliably produced in many differentspecies. Epitope tags are usually derived from viral genes, whichexplain their high immunoreactivity. Epitope tags include e.g. theV5-tag, MYC-tag, and HA-tag. These tags are particularly useful forwestern blotting, immunofluorescence and immunoprecipitationexperiments, although they also find use in protein purification.

Processing tags find many other usages, such as specific enzymaticmodification (such as biotin ligase tags) and chemical modification(FlAsH) tag. Often tags are combined to produce multifunctionalmodifications of the protein. Preferably, said marker is at least oneselected from the group consisting of

-   -   a radiolabel, preferably a radioactively labelled peptide or        protein    -   a fluorescent label, preferably a fluorescent peptide or        protein, and/or    -   an enzyme label, preferably a peroxidase.

This enumeration of potential marker payloads is by no meansrestrictive. According to another preferred embodiment, said drug is atleast one selected from the group consisting of

-   -   a cytokine    -   a radioactive agent    -   an anti-inflammatory drug    -   a toxin, and/or    -   a chemotherapeutic agent

This enumeration of potential drug payloads is by no means restrictive.As used herein, the term “cytokine” refers to small cell-signalingprotein molecules that are secreted by numerous cells and are a categoryof signaling molecules used extensively in intercellular communication.Cytokines can be classified as proteins, peptides, or glycoproteins; theterm “cytokine” encompasses a large and diverse family of regulatorsproduced throughout the body by cells of diverse embryological origin.In the present context, cytokines are for example meant to impair, oreven kill, pathogenic entity, e.g., a cancer cell.

As used herein, the term “radioactive agent” relates to an entity whichhas at least one atom with an unstable nucleus, and which is thus proneto undergo radioactive decay, resulting in the emission of gamma raysand/or subatomic particles such as alpha or beta particles, which have acell killing effect. In the present context, radioactive agents aremeant to impair, or even kill, pathogenic entity, e.g., a cancer cell.

As used herein, the term “anti-inflammatory drug” relates to compoundsthat reduce inflammation. This can be, e.g., steroids, just likespecific glucocorticoids (often referred to as corticosteroids), whichreduce inflammation or swelling by binding to glucocorticoid receptors.The term further encompasses non-steroidal anti-inflammatory drugs(NSAIDs), which counteract the cyclooxygenase (COX) enzyme. On its own,COX enzyme synthesizes prostaglandins, creating inflammation. In whole,the NSAIDs prevent the prostaglandins from ever being synthesized,reducing or eliminating the pain. The term further encompasses ImmuneSelective Anti-Inflammatory Derivatives (ImSAIDs), which are a class ofpeptides that alter the activation and migration of inflammatory cells,which are immune cells responsible for amplifying the inflammatoryresponse.

As used herein, the term “toxin” relates to a molecule which is toxic toa living cell or organism. Toxins may be peptides, or proteins orpreferably small molecular weight compounds, that are meant to impair,or even kill, pathogenic entity, e.g., a cancer cell. Toxins, as meantherein, encompass, in particular, cellular toxins. Preferably, saidtoxin is a small molecular toxin, i.e., having a molecular weight of≤2500 Da.

As used herein, the term “chemotherapeutic agent” relates to moleculesthat have the functional property of inhibiting a development orprogression of a neoplasm, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis or angiogenesis is frequently a property of anti-cancer orchemotherapeutic agents. A chemotherapeutic agent may be a cytotoxic orchemotherapeutic agent. Preferably, said chemotherapeuic agent is asmall molecular weight cytostatic agent, which inhibits or suppressesgrowth and/or multiplication of cancer cells.

Conjugating cytokines, radioactive agents, toxins or chemotherapeuticagents to an immunologand can help to reduce side effects and risksrelated to their administration, because

a) the immunoligand directs the conjugate to a specific site, e.g., to apathogenic entity, e.g., a cancer cell where the payload effects itstoxic function. Thus, the systemic toxicity of the payload is reduced,while the local concentration of the latter at the site of action isincreased, thus provding a better efficacy while side effects arereduced.

b) it can be provided that the conjugate is internalized by thepathogenic entity, in such way that after internalization, the payloadis released and only then develops its desired cytotoxic function, i.e.,without affecting the surrounding cells or tissue.

The following table is a non restrictive list of potentialtargets/antigens (1^(st) column) and examples for existing immunoligandstargeting the former (2^(nd) column). The 3^(rd) column shows a nonrestrictive list of potential toxins, cytokines or chemotherapeuticagents. Note that the examples from the 1^(st) and the 3^(rd) column canbe combined with one another ad libitum, while hundereds of furthertargets and payloads exist. Respective target/payload combinations notexplicitly mentioned in the table are encompassed by the scope of thepresent invention.

example of an existing target/antigen immunoligand payload EndothelialGrowth factor Cetuximab Maytansinoides, e.g. receptor (EGFR) Mertansine,Ansamitocin′ Ravtansin, DM4, DM1 CD20 Rituximab, Ibritumomab,Calicheamicins, e.g. Tositumomab (mAb) Ozogamicin CD44 Doxorubicin MUC1Cantuzumab (mAb) bacterial Pseudomonas exotoxin PE38 CD30 Brentuximab(mAb) Monomethyl Auristatin F (MMAF); Monomethyl Auristatin E (MMAE)CD22 inotuzumab (mAb) Pyrrolobenzodiazepine (PBD) transmembraneGlembatumumab (mAb) Interleukin-10 (IL10) (anti- glycoprotein NMBinflammatory) (GPNMB) CD56 Lorvotuzumab (mAb) Diphtheria toxin CanAghuC242 (mAb) Tumor necroris factor (TNF) luteinizing hormone [D-Lys(6)]LHRH RNase releasing hormone (LHRH) receptor Prostate-specific membraneYttrium⁹⁰ antigen (PSMA) CD74 Milatuzumab (mAb) Iodine¹³¹ CD70Lutetium¹⁷⁷ AGS-16 Cyclosporine Integrin Methotrexate CD19 Taxanes,e.g., Paclitaxel or Docetaxel Nectin-4 Interleukin 2 receptorInterleukin-2 (Proleukin) CD3 UCHT1 (mAb) extra domain B of L19-SIP(scFv fused in fibronectin with the constant domain CH4 SLAMF7 (CD319)Elotuzumab (mAb) SDC1 Indatuximab (mAb) Her-2/neu Trastuzumab (mAb) CD33Gemtuzumab (mAb)

According to yet another embodiment of the present invention, theimmunoligand comprises at least two subunits each being conjugated to apayload.

Preferably, at least two different payloads can be conjugated to the atleast two subunits. This option provides a versatile toolbox with whicha large variety of different immunoligand-payload constructs can becreated. For example, a bispecific dual-domain immunoligand can beconjugated with two different payloads, for example one marker and onetoxin.

Preferably the at least two different payloads are toxic payloadsinterfering with one or more cellular pathways.

Such embodiment can be accomplished, e.g., by conjugating the twodifferent payloads to each the 2 light chains of a full-length antibody,and to the 2 heavy chains of a full length antibody, respectively, byutilizing two different sortase enzymes, recognizing different sortaserecognition motifs, plus an antibody that contains different C-terminalmodifications at heavy and light chains comprising the respectiverecognition motifs for said different sortase enzymes.

In such way, an Antibody Drug Conjugate can be created which is composedof each two full-length Ig light chains and Ig heavy chains, containingdifferent payloads covalently attached to said heavy and light chains.

Such embodiment results, preferably, in the synchronous conjugation ofthe at least two subunits for the generation of immunoligand payloadswith equal payload conjugation to each of said subunits.

According to another preferred embodiment, said immunoligand with atleast two subunits is being conjugated with at least 80% efficiency perconjugation site.

According to yet another preferred embodiment, said immunoligand with atleast two subunits contains a peptide spacer sequence of at least twoamino acids, preferably 2-5 amino acids, appended to the C-termini of atleast one of the two subunits

This approach results, advantageously, in synchronous conjugation of theat least two subunits for the generation of immunoligand payloads withequal payload conjugation to each of said subunits.According to anotherembodiment of the present invention, the method allows astoichiometrically defined relationship between immunoligand andpayload. According to this embodiment, a strict quantitativerelationship between immunoligand and payload can be provided, thusimproving the reproducibility and the overall performance of therespective immunoligand/payload conjugate particularly for clinicaland/or therapeutic applications. This is accounted for by the sequence-and/or site specificity of the transpeptidase used.

According to a particularly prefered embodiment said stoichiometricallydefined relationship between immunoligand and payload is achieved byremoval of partially reacted C-terminally modified immunoligandsubstrate. Such removal can, for example, be carried out via affinitypurification. Said approach results, preferably, in a homogeneous drugto immunoligand ratio.

Preferably, said removal is carried out by affinity purification usingan affinity tag positioned C-terminal to the transpeptidase recognitionmotif or domain. Standard methods known to the skilled person can beused for this purpose, e.g., HIS tag, CBP tag, CYD (covalent yetdissociable NorpD peptide) tag, Strep II tag, FLAG tag, HPC (heavy chainof protein C) tag, and the GST and MBP protein fusion tags.

According to another embodiment of the present invention, the methodallows a site-specific conjugation of a payload to the immunoligand.According to this embodiment, it is ensured that the conjugation processdoes not interfere with the activity of the immunoligand, or thepayload, itself, thus improving the reproducibility and the overallperformance of the respective immunoligand/payload conjugateparticularly for clinical and/or therapeutic applications. This isaccounted for by the sequence- and/or site specificity of thetranspeptidase used. Other than with conventional binding chemistry,which is not site specific in most cases, or has limited sitespecificity (e.g, when the payload is conjugated to a free amino group,like in Arg, Lys, Asn or Gln), the binding site can thus be exactlydetermined, so that the characterizing features of the immunoligand(e.g., target specificity) or the payload (e.g., toxicity) are notaffected.

The invention further provides an immunoligand/payload conjugateobtained with a method according to the above-mentioned embodiments.

Preferably, said immunoligand/payload conjugate is selected from thegroup consisting of an antibody/drug conjugate, and/or anantibody/marker conjugate.

The invention further provides the use of an immunoligand/payloadconjugate according to the above mentioned embodiments for

-   -   in vitro or in vivo diagnosis of a given pathologic condition    -   in vitro or in vivo prediction or prognosis with respect to a        given pathologic condition    -   the treatment of a human or animal subject suffering from or        being at risk of developing a given pathologic condition, and/or    -   research and/or development purposes

Preferably, said pathologic condition is at least one selected from thegroup consisting of

-   -   Neoplastic disease    -   Autoimmune disease    -   Neurodegenerative disease, and/or    -   Infectious disease

In all these cases, the immunoligand/payload conjugate according to theinvention can have beneficial effects, e.g, by directing the latter to aspecific site, e.g., a cancer cell, a site of neuropathology, or a siteof an autoimmune reaction.

The payload, e.g., a toxin, a chemotherapeutic agent, a cytokine or adrug is delivered at said site, e.g., to deplete a cancer cell, to actanti-proliferatively on a cancer cell, to dissolve a plaque, to inhibitautoantibodies, and the like.

In all these cases, the immunoligand/payload conjugate according to theinvention can have beneficial effects, e.g, by directing the latter to aspecific site, e.g., a cancer cell, where the payload, e.g. a toxin or achemotherapeutic agent, is delivered, e.g., to deplete a cancer cell, toact anti-proliferatively on a cancer cell.

Thus, the systemic toxicity of the toxin or the chemotherapeutic agentis reduced, while the local of the latter at the site of action isincreased, thus provding a better efficacy while side effects arereduced. Further, a respective signalling cascade can be inhibited bythe binding of the immunoligand. In case the payload is a marker thelatter can thus be used to mark a specific site, e.g., a cancer cellcharcterized by a given surface antigen detected by the immunoligand,for diagnosis.

The site-specifity of the conjugating process ensures a highreproducibility and overall performance of the respectiveimmunoligand/payload conjugate particularly for clinical and/ortherapeutic applications.

The term “neoplastic disease”, as used herein, refers to an abnormalstate or condition of cells or tissue characterized by rapidlyproliferating cell growth or neoplasm. In a more specific meaning, theterm relates to cancerous processes, e.g., tumors and/or leukemias.

The term “neuropathological diseases” encompasses, among others,neurodegenerative diseases, neuroinflammatory diseases or seizuredisorders.

Neurodegenerative diseases are characterized by progressive loss ofstructure or function of neurons, including death of neurons. Manyneurodegenerative diseases including Parkinson's, Alzheimer's,Huntington's, Amyotrophic lateral sclerosis and Multiple Sclerosis occuras a result of neurodegenerative processes. There are many parallelsbetween different neurodegenerative disorders including atypical proteinassemblies as well as induced cell death. Neurodegeneration can furtherbe found in many different levels of neuronal circuitry ranging frommolecular to systemic.

The terms “Neurodegenerative diseases” and “Neuroinflammatory diseases”have a partially overlapping scope. Inflammatory responses are ahallmark of neurodegenerative disease and participate, or contribute,through different mechanisms in the neuronal cell death. The tryptophancatabolism along the Kynurenine pathway (KP) represents one of thesemechanisms.

Seizure disorders are brain disorders which are characterized byabnormal signaling between brain cells. Seizure disorders can affectpart of the brain (Partial seizures) or the entire brain (Generalizedseizures). The most prominent Seizure disorder is epilepsy.

The term “Autoimmune disease”, as used herein, encompassesorgan-specific autoimmune diseases, in which an autoimmune response isdirected against a single tissue, such as Crohn's disease and ulcerativecolitis, Type I diabetes mellitus, myasthenia gravis, vitiligo, Graves'disease, Hashimoto's disease, Addison' s disease and autoimmunegastritis and autoimmune hepatitis. The term also encompasses non-organspecific autoimmune diseases, in which an autoimmune response isdirected against a component present in several or many organsthroughout the body.

Such autoimmune diseases include, for example, rheumatoid arthritis,disease, systemic lupus erythematosus, progressive systemic sclerosisand variants, polymyositis and dermatomyositis .

Additional autoimmune diseases include pernicious anemia including someof autoimmune gastritis, primary biliary cirrhosis, autoimmunethrombocytopenia, Sjogren's syndrome, multiple sclerosis and psoriasis.One skilled in the art understands that the methods of the invention canbe applied to these or other autoimmune diseases, as desired.

The term “infectious disease” as used herein, includes, but is notlimited to any disease that is caused by an infectious organism.Infectious organisms may comprise viruses, (e.g., single stranded RNAviruses, single stranded DNA viruses, human immunodeficiency virus(HIV), hepatitis A, B, and C virus, herpes simplex virus (HSV),cytomegalovirus (CMV) Epstein-Barr virus (EBV), human papilloma virus(HPV)), parasites (e.g., protozoan and metazoan pathogens such asPlasmodia species, Leishmania species, Schistosoma species, Trypanosomaspecies), bacteria (e.g., Mycobacteria, in particular, M. tuberculosis,Salmonella, Streptococci, E. coli, Staphylococci), fungi (e.g., Candidaspecies, Aspergillus species), Pneumocystis carinii, and prions.

The invention further provides a low molecular-weight payload modifiedwith a Gly_(n)-modification, wherein, n>1, preferably n=3 or n=5.

As used herein, the term “Gly_(n)-modification” means that an oligo- orpolypepide consisting of n Glycin residues has been added to saidpayload. As used herein, the term “low molecular-weight payloadcompound” shall encompass payloads that have a molecular weight of 2500Da or less.

Said payload is, preferably, at least one selected from the groupconsisting of

-   -   a marker,    -   a processing tag, and/or    -   a drug.

Said marker is at least one selected from the group consisting of

-   -   a radiolabel, preferably a radioactively labelled peptide or        protein    -   a fluorescent label, preferably a fluorescent peptide or        protein, and/or    -   an enzyme label, preferably a peroxidase.

Said drug is at least one selected from the group consisting of

-   -   a cytokine    -   a radioactive agent    -   a toxin, and/or    -   a chemotherapeutic agent

As disussed above already, said toxin is preferably a small moleculartoxin, i.e., having a molecular weight of ≤2500 Da. Preferably, saidtoxin is at least one selected from the group constisting of

-   -   Maytansine    -   Monomethyl auristatin, and/or    -   Alpha-amanitin

or derivatives of the former. Examples for such Gly_(n)-modified toxionsare shown in structures 1 to 9 of FIG. 14A-14C

The invention further provides the use of a glycine-modified lowmolecular-weight payload for conjugation thereof to an immunoligand.

Preferably, and as mentioned above, the conjugation is atranspeptidease-mediated conjugation, preferably with a sortase and/or asplit intein. Likewise preferably, the immunologand is an antibody.

Preferably, said immunoligand is an antibody. In such way, an antibodydrug conjugate (ADC) can be provided.

Preferably, the immunologand-payload conjugation reaction is performedin crude cell culture supernatant. This means that, preferably, theconjugation reaction may take place with unpurified or only partiallypurified components.

Experiments and Figures

While the invention has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive; theinvention is not limited to the disclosed embodiments. Other variationsto the disclosed embodiments can be understood and effected by thoseskilled in the art in practicing the claimed invention, from a study ofthe drawings, the disclosure, and the appended claims. In the claims,the word “comprising” does not exclude other elements or steps, and theindefinite article “a” or “an” does not exclude a plurality. The merefact that certain measures are recited in mutually different dependentclaims does not indicate that a combination of these measures cannot beused to advantage. Any reference signs in the claims should not beconstrued as limiting the scope.

All amino acid sequences disclosed herein are shown from N terminus toC-terminus; all nucleic acid sequences disclosed herein are shown5′->3′.

EXAMPLE 1 Cloning of Expression Vectors and Expression of a CD19Monoclonal Antibody with C-Terminal LPETG Sortase Tag and Additional6×-His and StrepII Affinity Purification Tags

In order to perform the C-terminal conjugation of a payload to anantibody, first a recombinant antibody needs to be expressed thatcontains C-terminal modifications, including a recognition motif, e.g.for sortase A of Staphylococcus aureus.

For this, first ORFs for heavy and light chains of an anti-human CD19specific antibody can be gene synthesized, e.g. at contract researchorganizations (CROs) offering such gene synthesis services, like e.g.Genscript (www.genscript.com, Piscataway, N.J., USA). As an example, theheavy and light chain sequences of a humanized anti-human CD19 antibodyhBU12 can be found in patent U.S. Pat. No. 8,242,252 B2 under Seq 53(variant HF) and Seq 58 (variant LG). The V_(H) and V_(L) regions ofthis anti-human CD19 antibody are as follows:

SEQ ID NO 1 (V_(H) coding region of humanized anti-human CD19 antibody hBU12):ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTGTCCATTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGAGTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTTTAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGCTAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCC TTGTCACAGTCTCCTCAThis translates to the following amino acid sequence (SEQ ID NO 2):MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSSEQ ID NO 3 (V_(L) coding region of humanized anti-human CD19 antibody hBU12)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTCTCTCCAGGGGAAAGGGCTACCCTGAGCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTCCTGATTTATGACACATCCAAAGTGGCTTCTGGTATTCCAGCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTACACTCACAATCAGCAGCCTGGAGCCAGAGGATGTTGCTGTCTATTACTGTTTTCAGGGGAGTGTATACCCATTCACTTTTGGCCAAGGGACAAAGTTGGAAATCAAA This translates to the following amino acidsequence (SEQ ID NO 4):MKLPVRLLVLMFWIPASSSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDVAVYYCFQGSVYPFTFGQGTKLEIK

These sequences can be fused to human IgG_(i) constant heavy andconstant light chain regions containing additional C-terminal tags, inorder to realize the method disclosed herein.

In order to realize the invention, the human constant IgG1 heavy chainregion can be synthesized with additional 3′-codons, encoding an LPETGStaphylococcus aureus sortase A recognition tag, followed by a 6×His tag(HHHHHH), a MYC-tag (EQKLISEEDL) and a strep II tag (WSHPQFEK) resultingin a sequence, which is as follows:

SEQ ID NO 5 (human IgG1 heavy chain constant coding region with in-frame 3′ extension encoding an LPETG sortase tag, an 6xHis tag and a strepII tag):AGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAACTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAGTAGThis translates to the following amino acidsequence (SEQ ID NO 6, amino acids of the tags are underlined):STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGHHHHHHGEQKLISEED LGWSHPQFEK•

Furthermore, the human constant IgG1 kappa light chain region can besynthesized with additional 3′-codons, encoding an LPETG Staphylococcusaureus sortase A recognition tag, followed by a 6×His tag and a strep IItag (WSHPQFEK) resulting in a sequence, which is as follows:

SEQ ID NO 7 (human IgG1 kappa light chain constantcoding region with in-frame 3′ extension encodingan LPETG sortase tag, an 6xHis tag, a Myc tag, and a strepII tag):ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTCTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCC AGTTCGAGAAGTAGThis translates to the following amino acidsequence (SEQ ID NO 8, amino acids of the tags are underlined):TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSFNRGECLPETGHHHHHHGEQKLISEEDLGWSHPQFEK•

The complete coding regions for LPETG sortase tag, 6×His and streplltagged heavy and light chains of the humanized anti-human CD19 antibodyhBU12 are then as follows:

SEQ ID NO 9 (Complete human IgG1 V_(H)-C_(H) heavy chaincoding region for hBU12 with C-terminal LPETGsortase tag, 6xHis tag, Myc tag, and a strepII tag):ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTGTCCATTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGAGTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTTTAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGCTAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCCTCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAACTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAG TAGThis translates to the following amino acidsequence (SEQ ID NO 10, amino acids of the tags are underlined):MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYILPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGHHHHHHGEQKLISEEDLGWSHPQFE  K•SEQ ID NO 11 (Complete human IgG1 V_(L)-C_(L) kappachain coding region for hBU12 with C-terminalLPETG sortase tag, 6xHis tag, Myc tag, and a strepII tag):ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTCTCTCCAGGGGAAAGGGCTACCCTGAGCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTCCTGATTTATGACACATCCAAACTGGCTTCTGGTATTCCAGCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTACACTCACAATCAGCAGCCTGGAGCCAGAGGATGTTGCTGTCTATTACTGTTTTCAGGGGAGTGTATACCCATTCACTTTTGGCCAAGGGACAAAGTTGGAAATCAAAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTCTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAGTAGThis translates to the following amino acidsequence (SEQ ID NO 12, amino acids of the tags are underlined):MKLPVRLLVLMFWIPASSSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDVAVYYCFQGSVYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGHHHHHHGEQKLIS EEDLGWSHPQFEK•

The coding regions for the heavy and light chains of the anti-human CD19specific antibody as disclosed in SEQ ID NOs 9 and 11, respectively, canthen be synthesized with flanking restriction enzyme sites (e.g. HindIIIand NotI) such that they can be cloned into a standard mammalianexpression vector, such as pCDNA3.1-hygro (+) (Invitrogen), by standardmolecular biology methods known in the art.

The complete DNA sequence of pCDNA3.1-hygro (+)-IgH chain expressionvector for the tagged hBU12 anti-human CD19 antibody will be as follows:

SEQ ID NO 13 (coding region of human IgG1 V_(H)-C_(H )heavy chain for hBU12 with C-terminal LPETG sortase tag, 6xHis tag and a strepII tag underlined, andHindIII and NotI cloning sites shaded):GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC

AGGTGTCCATTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGAGTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTTTAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGCTAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCCTCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAACTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGA

GCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

The complete DNA sequence of pCDNA3.1-hygro (+)-IgL chain expressionvector for the tagged hBU12 anti-human CD19 antibody will be as follows:

SEQ ID NO 14 (coding region of human IgG1 V_(L)-C_(L )kappa light chain for hBU12with C-terminal LPETG sortase tag, 6xHis tag, Myc tag, and a strepII tagunderlined, and HindIII and NotI cloning sites shaded):GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC

TGCTTCCAGCAGTGAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTCTCTCCAGGGGAAAGGGCTACCCTGAGCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTCCTGATTTATGACACATCCAAACTGGCTTCTGGTATTCCAGCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTACACTCACAATCAGCAGCCTGGAGCCAGAGGATGTTGCTGTCTATTACTGTTTTCAGGGGAGTGTATACCCATTCACTTTTGGCCAAGGGACAAAGTTGGAAATCAAAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTCTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCG

CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

These constructs allow upon transfection into mammalian cells, likee.g.—but not limited to —CHO cells, that are typically used forrecombinant antibody expression, the expression of the anti-human CD19specific humanized antibody hBU12 with C-terminal additions of a sortaseA tag, a 6×His tag, a Myc tag, and a strepII tag at both the IgH and IgLchains.

EXAMPLE 2 Cloning of Expression Vectors for Monoclonal Antibody withC-Terminal N-Intein Domain of Ssp GyrB 11 Split-Intein with AdditionalC-Terninal 6×His and strepII Affinity Purification Tags

Similar to the design of expression cassettes and vectors ofStaphylococcus aureus sortase A tagged IgG1 heavy and light chains, thecoding regions for a C-terminal fusion of N-intein domain of Ssp GyrB 11split-intein to either the IgH and IgL chain can be designed as follows,in order to gene synthesize the genes by a qualified CRO (e.g. Genscript(www.genscript.com, Piscataway, N.J., USA), with the same elements forthe anti-human CD19 antibody as disclosed further above.

The 150 amino acid sequence of the N-intein domain of Ssp GyrB 11split-intein can be found in a publication by Appleby et al. (2009), andis as follows:

SEQ ID NO 15 (N-intein domain of Ssp GyrB 11 split-intein):CFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGSIGVEKIINARKTKTNAKVIKVTLDNGESIICTPDHKFMLRDGSYKCAMDLTLDDSLMPLHRKISTTEDSGHMEAVLNYNHRIVNIEAVSETIDVYDIEVPHTHNFA LAS

Reverse translation of that amino acid sequence with mammalian codonusage will result in the coding sequence for the N-intein domain of SspGyrB 11 split-intein as follows:

SEQ ID NO 16 (endocing sequence for N-inteindomain of Ssp GyrB 11 split-intein):TGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGCCAGC

With this sequence information at hand, the complete IgG1 heavy chaincoding region for anti-human CD19 antibody hBU12 with C-terminalextension, comprising the N-intein domain of Ssp GyrB 11 split-intein,followed by a 6×His-tag and a strepII tag can be designed as disclosedin SEQ ID NO 17 below:

ATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGTCCAGTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGAGTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTTTAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGCTAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCCTCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGCCAGCCACCATCACCATCACCATGGCTGGAGCCACCCCCAGTTCGAGAAGTAGThis translates to amino acid sequence SEQ ID NO 18 (amino acids of the N-intein domain are underlined, 6xHis tag and strepII tag are shaded):MNFGLRLIFLVLTLKGVQCQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKCFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGSIGVEKIINARKTKTNAKVIKVTLDNGESIICTPDHKFMLRDGSYKCAMDLTLDDSLMPLHRKISTTEDSGHMEAVLNYNHRI

Likewise, a complete IgGI kappa light chain coding region for anti-humanCD19 antibody hBU12 with C-terminal extension, comprising the N-inteindomain of Ssp GyrB 11 split-intein, followed by a 6×His-tag and astrepII tag can be designed as disclosed in SEQ ID NO 19 below:

ATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGTCCAGTGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAAGTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACIGTCAGCAAAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGCCAGCCACCATCACCATCACCATGGCTGGAGCCACCCCCAGTTCGAGAAGTAGThis translates to amino acid sequence SEQ ID NO 20 (amino acids of theN-intein domain are underlined, 6xHis tag and strepII tag are shaded):MNFGLRLIFLVLTLKGVQCDIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGSIGVEKIINARKTKTNAKVIKVTLDNGESIICTPDHKFMLRDGSYKCAMDLTLDDSLMPLHRKISTTEDSGHMEAVLNYNHRIVNIEAVSETID

The coding regions for the N-intein modified heavy and light chains ofthe anti-human CD19 specific antibody as disclosed in SEQ ID NOs 17 and19, respectively, can then be synthesized with flanking restrictionenzyme sites (e.g. HindIII and NotI) such that they can be cloned into astandard mammalian expression vector, such as pCDNA3.1-hygro (+)(Invitrogen), by standard molecular biology methods known in the art.

The complete DNA sequence of pCDNA3.1-hygro (+)-IgH chain expressionvector for the N-intein tagged hBU12 anti-human CD19 antibody is then asfollows:

SEQ ID NO 21 (coding region of human IgG1 V_(H)-C_(H )heavy chain for hBU12 with C-terminal N-intein domain of Ssp GyrB S11 split intein, followed by 6xHis tagstrepII tag (underlined), and HindIII and NotI cloning sites (shaded)):GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC

AGGCGTCCAGTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGAGTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTTTAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGCTAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCCTCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGCCAGCCACCATCACCATCACCATGGCTGGAGCCACCCCCAGT

TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

The complete DNA sequence of pCDNA3.1-hygro (+)-IgL chain expressionvector for the Ssp GyrB S11 N-intein domain tagged hBU12 anti-human CD19antibody will be as follows:

SEQ ID NO 22 (coding region of human IgG1 V_(L)-C_(L )kappa light chain for hBU12with C-terminal Ssp GyrB S11 N-intein domain, 6xHis tag and a strepII tagunderlined, and HindIII and NotI cloning sites shaded):GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC

AGGCGTCCAGTGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAAGTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGIGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGC

CCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

These pcDNA3.1-hygro(+) based expression vectors disclosed in SEQ ID NOs21 and 22 allow upon transfection into mammalian cells, like e.g. butnot limited to CHO cells, that are typically used for recombinantantibody expression, the expression of the anti-human CD19 specifichumanized antibody hBU12 with C-terminal N-intein domain fused, followedby a 6×His tag and a strepll tag at both the IgH and IgL chains.

EXAMPLE 3 Cloning and Expression of Recombinant Sortase A Enzyme fromStaphylococcus aureus

The ORF of Sortase A from Staphylococcus aureus is published in Genbankand can be found under entry: AF162687.1. The aa-sequence in that recordreads is shown as SEQ ID NO 23 (amino acid sequence of sortase A fromStaphylococcus aureus):

MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVKThe corresponding nucleotide sequence in thisGenbank entry is provided as SEQ ID NO 24:ATGAAAAAATGGACAAATCGATTAATGACAATCGCTGGTGTGGTACTTATCCTAGTGGCAGCATATTTGTTTGCTAAACCACATATCGATAATTATCTTCACGATAAAGATAAAGATGAAAAGATTGAACAATATGATAAAAATGTAAAAGAACAGGCGAGTAAAGATAAAAAGCAGCAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTATATTGAAATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACCAGCAACACCTGAACAATTAAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAATCACTAGATGATCAAAATATTTCAATTGCAGGACACACTTTCATTGACCGTCCGAACTATCAATTTACAAATCTTAAAGCAGCCAAAAAAGGTAGTATGGTGTACTTTAAAGTTGGTAATGAAACACGTAAGTATAAAATGACAAGTATAAGAGATGTTAAGCCTACAGATGTAGGAGTTCTAGATGAACAAAAAGGTAAAGATAAACAATTAACATTAATTACTTGTGATGATTACAATGAAAAGACAGGCGTTTGGGAAAAACGTAAAATCTTT GTAGCTACAGAAGTCAAATAA

Technical information with respect to the expression of an enzymaticallyactive fragment of recombinant sortase A in E.coli, comprising aminoacids 60-205 with 6×His tag are disclosed in reference WO2007/108013A2.The coding region for a 6×His tagged version of Staphylococcus aureussortase A (aa60-205) is provided below as SEQ ID NO 25:

ATGCAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTATATTGAAATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACCAGCAACACCTGAACAATTAAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAATCACTAGATGATCAAAATATTTCAATTGCAGGACACACTTTCATTGACCGTCCGAACTATCAATTTACAAATCTTAAAGCAGCCAAAAAAGGTAGTATGGTGTACTTTAAAGTTGGTAATGAAACACGTAAGTATAAAATGACAAGTATAAGAGATGTTAAGCCTACAGATGTAGGAGTTCTAGATGAACAAAAAGGTAAAGATAAACAATTAACATTAATTACTTGTGATGATTACAATGAAAAGACAGGCGTTTGGGAAAAACGTAAAATCTTTGTAGCTACAGAAGTCAAACACCAT CACCATCACCATTAAThis translates to amino acid sequence SEQ ID NO 26:MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKHH HHHH•

The coding region for the 6×His tagged sortase A fragment ofStaphylococcus aureus, as provided in SEQ ID NO 25, can be cloned into astandard bacterial expression vector, like e.g. pET29 (Novagen), inorder to transform E. coli strain BL21(DE3) (Novagen) and to generate anE. coli clone that can be used for the bacterial production ofrecombinant sortase A according to standard methods known in the art. Inshort, E. coli BL21(DE3) transformed with pET29 expression plasmids forsortase A can be cultured at 37° C. in LB mediom with 50 μg/mL kanamycinuntil until an oD₆₀₀=0.5-0.8 is reached. IPTG can then be added to afinal concentration of 0.4 mM and protein expression can be induced forthree hours at 30° C. The cells can then be harvested by centrifugationand resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaClsupplemented with 1 mM MgCl2, 2 units/mL DNAseI (NEB), 260 nM aprotinin,1.2 μM leupeptin, and 1 mM PMSF). Cells can then be lysed by sonicationand clarified supernatant can then be purified on Ni-NTA agarosefollowing the manufacturer's instructions.

Fractions that are of >90% purity, as judged by SDS-PAGE, can then beconsolidated and dialyzed against Tris-buffered saline (25 mM Tris pH7.5, 150 mM NaCl), and the enzyme concentration can be calculated fromthe measured A₂₈₀ using the published extinction coefficient of 17,420M⁻¹ cm⁻¹.The above-mentioned protocol has been followed and ca. 20 mgof >90% pure recombinant enzymatically active fragment (of ca. 17 kD)sortase A of Staphylococcus aureus has been produced and the analysis ofthe recombinant protein by SDS-PAGE and Western blotting is disclosed inFIG. 7.

EXAMPLE 4 Expression and Purification of Sortase Tagged or N-InteinTagged Recombinant Antibodies in CHO Cells

a.) CHO cell expression: Expression of recombinant IgG1 antibodies fromthe expression constructs disclosed under Examples 2 and 3 can beachieved by transient transfection using e.g. commercially available CHOexpression systems, like the FreeStyle CHO system from Invitrogenfollowing the instructions of the FreeStyle CHO manual.

In brief, about 1 day prior to transfection, CHO cells shall be seededat 5-6×10⁶ cells/ml in FreeStyle CHO medium in shaker-flasks in order toexpand them at 120 rpm on an orbital shaker at 37° C. in a humidifiedincubator at 7.5% CO₂ atmosphere. The following day the cells can betransfected, when they reach a density of 1.2-1.5×10⁶/ml. Cells thenneed to be diluted to 1×10⁶ cells/ml. 30 ml of such a cell suspensionthen needs to be added to a 125 ml shake flask and 40 μg of 1:1 mixedIgH and IgL expression plasmid DNA is added to 600 μl OptiPro SF-medium(Invitrogen). At the same time, 40 μl of FreeStyle MAX transfectionreagent needs to be added to 600 μl OptiPro SF-medium, and both samplesneed to be gently mixed, and incubated for 10 min at room temperature toallow DNA-transfection reagent complexes to form. Then theDNA-transfection reagent mix can be added slowly to the 125 ml CHO cellculture from above and the transfected cells are then grown for up to 6days at 120 rpm on an orbital shaker at 37° C. in a humidified incubatorat 7.5% CO₂ atmosphere. Thereafter, cell culture supernatant can becollected and analyzed for antibody expression titer by appropriatemethods known in the art (ELISA, Luminex, etc.).

b.) Protein A purification: Protein A purification of recombinantantibodies from the CHO cell supernatant can be performed withcommercially available protein A sepharose columns (Thermo Fisher,Pierce) according to instructions from the manufacturer.

In brief, cleared cell culture supernatant is run over a protein Acolumn of appropriate size and capacity equilibrated with PBS. Residualmedium is washed with PBS and eventually bound IgG can be eluted withlow pH buffer, like 0.1 M citric acid-NaOH, pH 3.0. Eluted IgG should beneutralized immediately with 1/10th volume of 1M Tris/Cl, pH7.4.Combined fractions containing IgG can then be dialized against PBS overnight at 4° C.

The protocols provided in Example 4 provide the skilled person in theart with the instruction to produce sufficient quantities of purified,recombinant antibodies from the constructs disclosed in Examples 1 and2.

EXAMPLE 5 Generation of Site-Specifically C-Terminally MMAE ToxicPayload Conjugated Monoclonal Antibodies by Sortase and Split-InteinMediated Transpeptidation

Monomethyl Auristatin A toxin coupled to a 5 amino acid glycine stretchand a 6 amino acid SSp GyrB Si i C-int split intein peptide according tothe formulas provided below, can be custom ordered from qualifiedchemistry CROs.

a.) Toxic MMAE payload conjugation of LPETG sortaseA motif taggedrecombinant IgG antibodies

Conjugation of 5 glycine amino acid modified MMAE toxic payload to LPETGsortase A tagged IgG1 antibody (that can be produced by followingExamples 1 and 4) can be achieved by mixing appropriate ratios of LPETGtagged IgG1 antibody with the glycine-modified MMAE toxin disclosed inFormula 1 (e.g. at 1:1 ratio and 50 μM concentration) and withrecombinant sortase A (production described in Example 3) (e.g. at 5 μMconcentration), and using physiologic incubation buffer, like e.g.; 5 mMTris/Cl, 15 mM NaCl, 6 mM CaCl₂, pH 8.0, and incubating at 37° C. to 40°C. for a minimum of 2 hours.

Efficiency of the conjugation can be monitored by analyzing the absenceof the 6×His tag and/or the strepII tag after stopping the reaction,e.g. by western-blot analysis or ELISA with anti-His-tag and/or antistrepII tag antibodies.

Completely conjugated product can be enriched by Nickel-NTA columns, orstreptactin column binding, which bind to the 6×His tag or strepll tag,respectively, which can only be present in incompletely reacted IgG1substrate. Final IgG-payload conjugate can eventually be purified usingprotein A purification as described above.

b.) Toxic MMAE payload conjugation of SSp GyrB Sll N-intein taggedrecombinant IgG antibodies

Conjugation of Ssp GyrB S11 C-intein amino acid modified MMAE toxicpayload to N-intein tagged IgG1 antibody (that can be produced byfollowing Examples 2 and 4) can be achieved by mixing appropriate ratiosof N-intein tagged IgG1 antibody with the C-intein amino acid-modifiedMMAE toxin disclosed in Formula 2 (e.g. at 1:10 or 1:25 ratio at 5 μMconcentration of the IgG antibody) using physiologic incubation buffer,like e.g.; 20 mM Tris/Cl, 250 mM NaCl, 1 mM EDTA, pH 8.5, and incubatingat room temperature or at 37° C. a minimum of 4 hours.

Efficiency of the conjugation can be monitored by analyzing the absenceof the 6×His tag and/or the strepII tag after stopping the reaction,e.g. by western-blot analysis or ELISA with anti-His-tag and/or antistrepII tag antibodies.

Completely conjugated product can be enriched by Nickel-NTA columns, orstreptactin column binding, which bind to the 6×His tag or strepII tag,respectively, which can only be present in incompletely reacted IgG1substrate. Final IgG-payload conjugate can eventually be purified usingprotein A purification as described above.

In summary, the Examples 1-5 disclosed above allow a person skilled inthe art to practice the invention of enzymatically conjugating a toxicpayload site-specifically to the C-terminus either using sortase Amediated or split-intein mediated transpeptidation.

EXAMPLE 6 Production of Trastuzumab with C-Terminal GS (Glycine-Serine)Linker, LPETG Sortase Motif and Additional 6×-His and Strep II AffinityPurification Tags on Either Heavy or Light Chain

Antibody expression constructs encoding monoclonal antibody Trastuzumab(Tras) heavy and light chains, either untagged (SEQ ID NOs: 31-34) orC-terminally tagged with GS (glycine-serine) linker, LPETG Sortase tag,6×His tag, and Strep II tag (SEQ ID NOs: 35-38) were generatedessentially as described in Example 1. Using these expressionconstructs, Tras-HC-GS-LHS and Tras-LC-GS-LHS (HC=heavy chain, LC=lightchain, GS=glycine-serine, LHS=LPETG-tag+6×His-tag+strepII-tag) wereproduced in CHO cells by co-transfection of the corresponding expressionconstructs. Tras-HC-GS-LHS is a Trastuzumab variant with an unmodifiedlight chain (SEQ ID NOs: 35-36), and a heavy chain C-terminally taggedwith GS (glycine-serine) linker, LPETG Sortase motif, 6×His-tag, andstrepII-tag (SEQ ID NOs: 33-34). Tras-LC-GS-LHS is a Trastuzumab variantwith an unmodified heavy chain (SEQ ID NOs: 31-32), and a light chainC-terminally tagged with GS linker, LPETG Sortase motif, 6×His-tag, andstrepII-tag (SEQ ID NOs: 37-38). CHO cell transfection and affinitypurification of antibodies by proteinA-sepharose chromatography was doneessentially as described in Example 4.

EXAMPLE 7 Sortase A-Mediated Conjugation of Heavy or Light Chain ofTrastuzumab with Gly5-Modified DM1 Toxin

Conjugation reactions containing Gly₅-modified DM1 toxin (ordered fromConcortis, San Diego, Calif., U.S., structure see FIG. 14 a.) and a 17kD recombinant sortase A fragment from Staphylococcus aureus (seeExample 3) were carried out with 10.5 mg of each monoclonal antibody(mAb) (see Example 6) in 1× Sortase buffer (25 mM Tris-HCl, pH8.2; 150mM NaCl; 7.5 mM CaCl₂), as shown in Table II, below. The Tras-HC-GS-LHSconjugation reaction was incubated at 25° C. for 2 h; the Tras-LC-GS-LHSconjugation reaction was incubated at 25° C. for 18h. Each reactionmixture was then passed over a Strep-Tactin® Sepharose columns (IBALife-Sciences, Göttingen, Germany). For this, lml of Strep-TactinAgarose was packed under gravity into a fitted column and equilibratedwith 2 column volumes of equilibration buffer (100 mM Tris-HCl, pH 8.0;150 mM NaCl; 1 mM EDTA). Each conjugation mixture was passed twice downthe same column using gravity flow (to increase residence time on theresin). The resin was washed with an additional column volume ofequilibration buffer to maximize conjugate yield and the pool thenapplied immediately to a protein A column. For this, a 1 ml Protein AHiTrap column was equilibrated with 10 column volumes of buffer (25 mMsodium phosphate pH 7.5). Each conjugation reaction was then applied toan equilibrated column and the column washed with a further 5 columnvolumes of buffer. Bound conjugate was eluted with 5 column volumes ofelution buffer (0.1M succinic acid, pH 2.8) with 1 column volumefractions collected (into tubes containing 25% v/v 1M Tris Base toneutralise the acid) and analysed for protein content. Proteincontaining fractions were pooled and formulated by G25 columnchromatography. For this, NAP 25 columns of an appropriate size for eachscale of manufacture were used to formulate the conjugates for long termstorage. The columns were equilibrated, loaded and eluted with 10 mMSodium Succinate pH 5.0, 100 mg/mL Trehalose, 0.1% % w/v Polysorbate 20(Formulation Buffer for Kadcyla® (T-DM1), marketed by Roche/Genentech)according to the manufacturer's instructions.

The Tras-HC-GS-LHS and Tras-LC-GS-LHS DM1-conjugate yields were,respectively, 8.0 mg (76.2%) and 5.9 mg (56.2%). The major processlosses occurred during Protein A and G25 purification, most probably asa result of peak cutting to ensure maximal concentration of the productfor each subsequent step or storage.

TABLE 2 Conjugation conditions for Tras-HC-GS-LHS and Tras-LC-GS-LHS:Final Reaction component HC LC concentration Tras-HC-GS-LHS (5.3 mg/ml)  1981 μl —  5 μM Tras-LC-GS-LHS (5.5 mg/ml) — 1911 μ1  5 μM H₂0 7775.25μl 7714 μl — G1y₅-DM1 (1 mM)   1400 μl 1400 μl 100 μM Sortase A (0.85mg/ml = ca.  43.75 μl  175 μl 0.156/0.625 μM 50 μM) 5× Sortase buffer*  2800 μl 2800 μl 1×

The drug loading was assessed by Hydrophobic Interaction Chromatography(HIC), and was performed on a TOSOH Butyl-NPR 4.6 mm×3.5 cm, 2.5 μmcolumn run at 0.8 mL/min with a 12 minute linear gradient between A−1.5M (NH₄)₂SO₄, 25 mM NaPi, pH=6.95±0.05 and B −75% 25 mM NaPi,pH=6.95±0.05, 25% IPA. The HIC profiles revealed that for both,Tras-HC-GS-LHS and Tras-LC-GS-LHS, there was no detectable unconjugatedmAb left, and a major fraction of each mAb was loaded with 2 drugs (seeFIG. 8).

EXAMPLE 8 In Vitro Toxicity Assay with Sortase A—MediatedTrastuzumab-DM1 Conjugates

Cytotoxicity of DM1-sortaseA-conjugated Tras-HC-GS-LHS andDM1-sortaseA-conjugated Tras-LC-GS-LHS was investigated and compared toKadcyla® (Roche/Genentech) using SKBR3 cells, a human breast cancer cellline overexpressing the cognate antigen of trastuzumab (Tras) HER-2/neu,and T47D-5R cells, a breast cancer cell line naturally expressing lowlevels of HER-2/neu, engineered to be devoid of cell surface HER-2/neu(Graus-Porta et al. (1995)). Cells were plated on 96 well plates in 100μl complete DMEM (10'000 cells per well). After one day incubation, 50μl medium was carefully removed from each well and replaced by 50 μl of3.5-fold serial dilutions of each ADC in complete DMEM, resulting in ADCconcentrations ranging from 20 μg/ml to 0.25 ng/ml. Each dilution wasdone in duplicates or triplicates. After 3 additional days incubation at37° C. in a humidified incubator at 5% CO₂ atmosphere, plates wereremoved from the incubator and equilibrated to room temperature. Afterapproximately 30 minutes, 100 μl CellTiter-Glo® Luminescent Solution(Promega, Cat.No G7570) was added to each well and, after shaking theplates at 450 rpm for 5 min followed by a 10 min incubation withoutshaking, luminescence was measured on a Tecan Infinity F200 with anintegration time of 1 second per well. All three ADCs were highlycytotoxic for the HER-2/neu overexpressing SKBR3 breast cancer cellline, but not for the HER-2/neu-negative T47D-5R breast cancer cell line(see FIG. 9). The EC₅₀ values for Her-2/neu positive breast cancer cellline SKBR3 were: Kadcyla®, 32.4 ng/ml; DM1-conjugated Tras-HC-GS-LHS,45.6 ng/ml; Tras-LC-GS-LHS, 51.4 ng/ml, and thus are within similarrange of potency in the in vitro tumor cell killing experiment.Conversely, no specific cellular toxicity was detectable with theHer-2/neu negative breast cancer cell line T47D-5R, demonstrating thefunctional equivalence of sortaseA, enzymatically conjugated ADC versustraditional, chemically conjugated ADC, when the comparison entails thesame targeting antibody and the same toxin (DM1) (FIG. 9). However, itappears that the lower drug-to antibody ratio of ca. 1.80 (deducted fromintergration of the DAR1 and DAR2 peaks in FIG. 8) for theTras-HC-GS-LHS and Tras-LC-GS-LHS sortase A-conjugated ADCs, as comparedto the DAR of ca. 3-4, reported for Kadcyla® does not translate into aproportionally different cellular cytotoxicity in the in vitro tumorcell killing assays (FIG. 9). This unexpected finding may be the resultof a more defined and site-specific toxin-antibody conjugation mediatedby sortase A in comparison to the less defined, stochastically,chemically conjugated Kadcyla®.

EXAMPLE 9 Optimization of Synchronization of SortaseA Mediated AntibodyHeavy Chain and Light Chain Payload Conjugation by Variation ofPeptide-Spacer Length Inserted Between C-Terminal End of Antibod HeavyChain and Light Chain and the SortaseA Recognition Motif

The influence of peptide-spacer length positioned between the C-terminusof antibody heavy or light chain and LPETG sortase A recognition motifwas investigated. For this, antibody heavy chain and light chainexpression constructs encoding chimeric CD30-specific mAb Ac10 heavy andlight chains (HC sequence derived from US 2008213289A1, Seq1, LCsequence derived from US 2008213289A1, Seq9), C-terminally modified withsequences comprising or not comprising a 2 amino acid GS(glycine-serine) spacer, and comprising a LPETG sortaseA recognitionmotif, and a strep-II purification tag (SEQ ID NOs: 39-46), have beencloned essentially according to instructions disclosed in Example 1.Using these expression constructs, mAbs Ac10-HC-GS-LHS/LC-GS-LHS andAc10-HC-LS/LC-LS were produced in CHO cells by co-transfection of thecorresponding plasmids. Ac10-HC-GS-LHS/LC-GS-LHS is an Ac10 variant withheavy and light chains modified at the C-termini of each HC and LC witha GS peptide spacer, a LPETG sortaseA motif, a 6×His tag, and a strep-IItag (SEQ ID NOs:39-42; Table 3). Ac10-HC-LS/LC-LS is an Ac10 variantwith heavy and light chains modified at the C-termini with LPETG Sortasemotif and strep-II tag without the 2-peptide GS linker (SEQ ID NOs:43-46; Table 3). CHO cell transfection and affinity purification ofantibodies by protein A-sepharose chromatography was done essentially asdescribed in Example 4.

To investigate efficiency of conjugation, serial dilutions of Sortase Awere used to conjugate penta-glycine-modified FITC (Gly₅-FITC, seeFormula 3 below).

For this, Gly₅-FITC was sortaseA conjugated to two Ac10 variants in 1×Sortase buffer (25 mM Tris-HCl, pH8.2; 150 mM NaCl; 7.5mM CaCl₂), asshown in Table 4. After 4 h at 42° C., reaction products were analyzedby denaturing, reducing SDS-PAGE gel electrophoresis, and FITC wasvisualized by placing the gels on a UV box (FIG. 10). Conjugation to theheavy chain was found to be highly efficient irrespective of thepresence absence of the GS-linker between heavy chain C-terminus andLPETG Sortase recognition motif. Unexpectedly, sortaseA mediatedconjugation to the light chain was significantly less efficient incomparison to sortaseA mediated heavy chain conjugation. Furthermore, itwas surprisingly found that coupling efficiency was dramaticallyaffected by the presence or absence of the 2 peptide GS (glycine-serine)spacer positioned between the C-terminus of the antibody light chainsand the LPETG sortaseA recognition motif. Whereas in the presence of theGS-linker, conjugation to the light chain took place with about 5-10×lower efficiency than to the heavy chain, it was about 50-100× lessefficient in the absence of a linker. Therefore, it was concluded thatincreasing the peptide spacer length between the light chain and theLPETG Sortase recognition motif might further improve conjugationefficiency.

Therefore, the influence of increasing the length of the peptide spacerbetween light chain and LPETG Sortase A recognition motif on conjugationefficacy was investigated next. Expression constructs encoding mAb Ac10light chains, C-terminally tagged with LPETG Sortase recognition motifand strep-II purification tag, with a 2 to 5 amino acid linker (SEQ IDNOs: 47-54), were generated essentially as described in Example 1. Usingthese expression constructs, mAbs Ac10-HC-LS/LC-GS-LS,Ac10-HC-LS/LC-GGS-LS, Ac10-HC-LS/LC-GGGS-LS and Ac10-HC-LS/LC-GGGGS-LSwere produced in CHO cells by co-transfection of the correspondingexpression constructs. In each of these antibodies, the heavy chain isC-terminally modified with an LPETG Sortase recognition motif and astrep-II purification tag (SEQ ID NOs: 43-44; Table 3). The light chainis C-terminally modified with an LPETG Sortase tag and strep-II tagcontaining either a GS, GGS, GGGS, or a GGGGS peptide spacer (SEQ IDNOs: 47-54; Table 3) in front of the LPETG motif. CHO cell transfectionand affinity purification of antibodies by protein A-sepharosechromatography was done essentially as described in Example 4.

To investigate conjugation efficiency, serial dilutions of Sortase Awere used to conjugate penta-glycine-modified FITC (Gly₅-FITC, seeFormula 3, above) to the four different Ac10 mAb variants in 1× Sortasebuffer (25 mM Tris-HCl, pH8.2; 150 mM NaCl; 7.5 mM CaCl₂), as shown inTable 5. After 4h at 42° C., reaction products were analyzed bydenaturing, reducing SDS-PAGE gel electrophoresis, and FITC wasvisualized by placing the gels on a UV box (FIG. 11). As expected,conjugation to the heavy chain was equally efficient in all fourantibody variants. In contrast, conjugation to the light chain wasimproved significantly by increasing peptide-spacer length.Significantly, with the longest peptide-spacer analyzed (GGGGS), lightchain conjugation efficiency was equally efficient in comparison toconjugation of the heavy chain, thereby allowing synchronous conjugationof heavy and light chains of an antibody C-terminally modified at bothheavy and light chain. It is concluded that this antibody format willfacilitate Sortase A-mediated production of homogeneous ADCs loaded with4 drugs per antibody (DAR4).

TABLE 3 C-terminally modified monoclonal antibody Ac10 variants producedHeavy Chain Light Chain Antibody modification SEQ ID NOs modificationSEQ ID NOs Ac10-HC-GS- GS-LPETG-G- 39, 40 GS-LPETG-G- 41, 42 LHS/LC-GS-HHHHHH-G- HHHHHH-G- LHS WSHPQFEK WSHPQFEK Ac10-HC- LPETG-G- 43, 44LPETG-G- 45, 46 LS/LC-LS WSHPQFEK WSHPQFEK Ac10-HC- LPETG-G- 43, 44GS-LPETG-G- 47, 48 LS/LC-GS-LS WSHPQFEK WSHPQFEK Ac10-HC- LPETG-G-43, 44 GGS-LPETG- 49, 50 LS/LC-GGS-LS WSHPQFEK G- WSHPQFEK Ac10-HC-LPETG-G- 43, 44 GGGS-LPETG- 51, 52 LS/LC-GGGS- WSHPQFEK G- LS WSHPQFEKAc10-HC- LPETG-G- 43, 44 GGGGS- 53, 54 LS/LC- WSHPQFEK LPETG-G- GGGGS-LSWSHPQFEK

TABLE 4 Conjugation conditions for mAbs Ac10-HC-GS-LHS/LC-GS-LHS andAc10-HC-LS/LC-LS Reaction component 1-8 9-16 Final concentrationAc10-HC-GS-LHS/LC-GS-LHS 10 —  5 μM (3.75 mg/ml = 25 μM)Ac10-HC-LS/LC-LS (3.75 mg/ml = ) — 10  5 μM 25 μM H₂0 20 20 — Gly₅-FITC(1 mM) 5 5 100 μM Sortase A (2× serial dil. of ca. 50 μM) 5 5 5 → 0.039μM 5× Sortase buffer 10 10 1×

TABLE 5 Conjugation conditions for mAbs Ac10-HC-LS/LC-GS-LS,Ac10-HC-LS/LC-GGS-LS, Ac10-HC-LS/LC-GGGS-LS and Ac10-HC-LS/LC-GGGGS-LS.Reaction component 1-7 8-14 15-21 22-28 Final conc. Ac10-HC-LS/LC-GS-LS10 — — —    5 μM (3.75 mg/ml = 25 μM) Ac10-HC-LS/LC-GGS-LS — 10 — —    5μM (3.75 mg/ml = 25μM) Ac10-HC-LS/LC-GGGS-LS — — 10 —    5 μM (3.75mg/ml = 25 μM) Ac10-HC-LS/LC-GGGGS-LS — — 10    5μM (3.75 mg/ml = 25 μM)H₂0 20 20 20 20 — G1y₅-FITC (1 mM) 5 5 5 5   100 μM Sortase A (2× serialdil. of 5 5 5 5 2.5 → ca. 25 μM) 0.039 μM 5× Sortase buffer 10 10 10 101×

EXAMPLE 10 Generation of Homogeneous ADC by StrepII-Tag AffinityPurification

Sortase A mediated conjugation with Gly₅-labeled vc-PAB-MMAE (seeFormula 1, Example 5) was performed with anti-CD30 antibody Ac10modified at the C-termini of either the heavy chains, or the lightchains with sequences comprising an LPETG sortase A motif and astrepThaffinity purification tag as provided in Table 6 below:

TABLE 6C-terminally modified antibody Ac10 with either HC or LC modificationHeavy Chain Light Chain Antibody modification SEQ ID NOs modificationSEQ ID NOs Ac10-HC-LS LPETG-G- 43, 44 none 29, 30 Ac-10-LC WSHPQFEKAc10-HC none 27, 28 GS-LPETG-G- 41, 42 Ac10-LC-GS- HHHHHH-G- LHSWSHPQFEK

The expression vectors encoding the Ac10 heavy or light chain sequencesof Table 4 have been constructed essentially as disclosed in Example 1.CHO cell transfection and affinity purification of antibodies by proteinA-sepharose chromatography was done essentially as described in Example4.

Sortase A mediated conjugation of heavy or light chaing sortase motiftagged anti-CD30 antibodies with Gly₅-labeled vc-PAB-MMAE (see Formula1, Example 5) was performed essentially according to the protocolprovided in Example 7.

As described further above in the detailed description of the invention,unreacted antibody will retain the C-terminal strep-II affinitypurification tag, which can be exploited to enrich fully reacted ADCwith DAR2. Analysis of the heavy chain sortase A conjugation withvc-PAB-MMAE toxin via hydrophobicity interaction chromatography (HIC)(FIG. 12, panel A), shows that the majority of the sortase-motifmodified heavy chains have been conjugated, but a certain percentage ofunreacted substrate (DAR0=drug to antibody ratio=zero), or partiallyreacted substrate (DAR1=drug to antibody ratio=1) was still detectableby HIC (FIG. 12, Panel A).

Therefore, the protein A purified vc-PAB-MMAE conjugate was passed 4times times over a StrepTactin® affinity column (IBA Sciences,Gottingen, Germany), essentially as described in Example 7, in order toremove unreacted or partially reacted sortase A-modified antibody. FIG.12, Panel B shows that upon several passages of the heterogeneousvc-PAB-MMAE antibody drug conjugate, completely reacted DAR2 ADCs(DAR2=drug to antibody ratio=2) could be highly enriched. Thisexperiment demonstrates the feasibility to utilize additional affinitypurification tags added C-terminally to the sortase A LPETG recognitionmotif to generate homogeneous ADC with a defined drugs per antibodyratio (here DAR2).

EXAMPLE 11 Synthesis of 5×Glycine-Modified Maytansine and Alpha-AmanitinToxins

In order to allow conjugation of two different payloads, preferablytoxic payloads to a single antibody, modified with different sortasemotifs at heavy and light chain C-termini, it is required to modify twodifferent toxins with glycine residues, preferably toxins with differentmode of actions, such that a cancer cell targeted with a dual payloadconjugated ADC, is attacked with via two different, potentiallysynergistic routes. The synthesis of two different glycine-modifiedtoxic payloads (here maytansine and alpha-amanitin) satisfying thisrequirement has been performed and is described herein.

11.1 Synthesis of Glycine-Modified Alpha-Amanitin:

30 mg alpha-amanitin (Structure 1) (Sigma-Aldrich, order # A2263) wasdissolved in 1 ml anhydrous DMSO. To this solution 19mgNH-Boc-amino-hexylbromide were added, followed by potassiumtert-butoxide (1M solution in THF, 35 μl). The reaction mixture wasstirred at room temperature for 6 h and more potassium tert-butoxide (1Msolution in THF, 20 μl) was added. The reaction was kept at roomtemperature for 16 h. Acetic acid (10 μl) was added and the crudemixture was purified by RP-HPLC directly (Sunfire C18 5μ 3 cm×10 cmcolumn, 50 mL/min, 5-50% acetonitrile/water 15 min gradient). Thedesired fraction was collected and lyophilized to give Structure 2 as awhite powder (15 mg), which was treated with TFA/DCM solution (1/1, v/v,1 ml) for 30 minutes at room temperature. The volatiles were removedunder reduced pressure to give Structure 3 as a slightly yellowish gum,which was used in the next step without further purification.

Fmoc-Gly5-OH (8 mg) was dissolved in anhydrous DMF (0.5 ml). HATU(Sigma-Aldrich, order # 445460) (6 mg) was added, followed by DIEA (10ml) (Sigma-Aldrich, order #496219). The mixture was agitated gently atroom temperature for 30s and then transferred to a solution of compound3 in DMF (0.5 ml). After 30 mins, LC/MS analysis showed that all ofcompound 3 was consumed. Piperidine (30 μl) was added and the progressof the reaction was monitored by LC/MS. Acetic acid was added toneutralize the reaction after 1 h and the mixture was purified byRP-HPLC (Sunfire C18 5μ 3 cm×25 cm column, 50 mL/min, 2-40%acetonitrile/water 30 min gradient). The fractions were pooled andlyophilized to give structure 5 as a white powder (12 mg). Analyticaldata for compound 5 is provided in FIG. 13, panel A).

11.2. Synthesis of Glycine-Modified Maytansine:

Maytansinol (0.6 g, 1.1 mmol) (Clearsynth Labs, Mumbai, India) wasdissolved in anhydrous THF (6 ml) and anhydrous DMF (3 ml) after which1,2 ml DIEA (Sigma-Aldrich, order #496219) was added. The solution wasplaced under argon atmosphere. Zinc triflate (1.2 g) and NMeAla NCA (0.7g) were added in one portion. The mixture was sonicated until the solidwas dissolved. The reaction mixture was stirred at room temperature for2 days and then diluted with ethyl acetate (100 ml). It was washed withsaturated NaHCO₃ (aq. solution, 2×50 ml) and brine (50 ml). The organiclayer was dried (over MgSO₄) and concentrated to give the crudemaytansinol 3-(S)-alpha-N-methylaminopropionate (8) which was useddirectly in the next step without further purification.

Fmoc-Gly5-OH (26 mg) was dissolved in anhydrous DMF (1 ml). HATU(Sigma-Aldrich, order # 445460) (19 mg) was added, followed by DIEA (18μL). The mixture was agitated gently at room temperature for 30s andthen transferred to a solution of compound 8 in THF (1 ml). After 30mins, LC/MS analysis showed that all compound 8 was consumed. Piperidine(40 μl) was added and the progress of the reaction was monitored byLC/MS. Ether (40 ml) was added to the reaction after 2 h and theprecipitated solid was collected and washed with ether. The crudecompound was purified by RP-HPLC (Sunfire C18 5μ 3 cm×10 cm column, 50ml/min, 10-60% acetonitrile/water 20 min gradient). The fractions werepooled and lyophilized to give compound 10 as a white powder (33 mg).Analytical data for compound 10 is provided in FIG. 13, Panel B.

Importantly, it is to be noted that in principle, any toxin can befunctionalized for sortase mediated enzymatic conjugation, if either 5glycines (as shown here), or any number of glycine residues greater orequal than one glycine, are attached to the toxins (see FIG. 14).

EXAMPLE 12 In Vivo Tumor Inhibition of Sortase A-ConjugatedTrastuzumab-DM1 in SKOV3 Ovarial Carcinoma Xenograft Models

5×10⁶ SKOV3 tumor cells in 200 μl PBS/Matrigel (1:1 ratio) wereimplanted subcutaneously into the left flanks of 5-6 weeks old femaleNMRI nude mice. Primary tumor volumes were monitored by calipering.After a mean tumor volume of 100-200 mm³ was reached, tumor-bearinganimals were randomized into 3 Groups according to tumor sizes (10animals per group). On the day of randomization (day 0) and on day 21,animals of Groups 1, 2 and 3 were injected intravenously with,respectively, 5 ml/kg PBS, 15 mg/kg Kadcyla®, or 15 mg/kg sortaseA-conjugated Trastuzumab-DM1. Tumor volumes were measured bi-weekly bycalipering (FIG. 15). The study was terminated after 39 days and animalswere euthanized according to accepted animal experimentation guidelines.

In the course of the study, tumors in control animals mock-injected withPBS grew steadily to a volume of approximately 600 mm³. In contrast,tumors in Kadcyla®-treated animals shrank and were essentiallyundetectable on day 39. Anti-tumor activity of Sortase A-conjugatedTrastuzumab-DM1 did not differ significantly from that of commerciallyavailable Kadcyla®, despite the fact that the sortase-conjugated T-DM1exhibited a lower drug to antibody ratio of approximately 2, incomparison of a reported DAR of 3.5 of Kadcyla®. In combination with thedata from Example 8, the results demonstrate that sortase conjugatedADCs, using identical antibody and toxin moiety, have comparable tumorkilling activity in comparison to commercially available chemicallyconjugated Kadcyla® in vitro and in vivo, albeit at lower drug toantibody ratio.

EXAMPLE 13 Sortase A-Mediated Conjugation in Crude CHO Cell Supernatant

The Trastuzumab variant Tras-HC-LS/LC-GGGGS-LS, consisting of heavychains C-terminally tagged with LPETG Sortase motif and Strep IIpurification tag (SEQ ID NOs: 055-056), and light chains C-terminallytagged with a 5 amino acid Gly₄-Ser spacer (GGGGS), LPETG Sortase motifand Strep II tag (SEQ ID NOs: 057-058), was produced in CHO cellsessentially as described in Example 4. The resulting serum-free crudecell supernatant contained approximately 157 mg/L Tras-HC-LS/LC-GGGGS-LSand was directly used for conjugation essentially as described inExample 9, by adding Sortase buffer, Gly_(s)-FITC, and serial dilutionsof Sortase A directly to the supernatant. In parallel,Tras-HC-LS/LC-GGGGS-LS purified by protein A affinity chromatography wasalso conjugated under otherwise identical conditions. After 4 hours at42° C., the reactions were analyzed by denaturing, reducing SDS-PAGE gelelectrophoresis. After visualizing FITC by placing the gel on a UV box,protein was stained using Coomassie Brilliant Blue (FIG. 16). The datashows the unexpected finding that Sortase A-mediated conjugation ofantibodies in crude cell culture supernatant was as efficient as that ofpurified antibody. Further, the conjugation reaction was highly specificand none of the protein contaminants present in crude CHO cellsupernatant were non-specifically conjugated. Together, these datasuggest that the robustness of the Sortase reaction may help facilitateADC manufacturing by allowing to perform drug conjugation directly afterproduction in CHO cells prior to purification and downstream processing.

FIGURE LEGENDS

FIG. 1: This figure illustrates the principle of the sortase A mediatedsite-specific payload conjugation to an immunoligand (or bindingprotein), which can be performed at the N-terminus of a protein (a), orat the C-terminus of the protein (b). In order to achieve N-terminalconjugation, the payload needs to contain a sortase penta-peptiderecognition motif (here LPXTG, the recognition motif of sortase A fromStaphylococcus aureus (X representing any of the 20 natural aminoacids), whereas the N-terminus of the immunologand/binding protein to belabeled needs to be expressed with an N-terminal extension of minimally3 glycine residues, here indicated as G_(n), (with n>2), that has a freeN-terminal amino group (here indicated by the smaller H₂N-symbol).Typically 3-5 glycines are used in order to modify a substrate forsortase-mediated conjugation. Addition of recombinant sortas A enzymefrom Staphylococcus aureus, as indicated here, then catalyzes thebreakage of the peptide bond between the T and the C-terminal G residuein the LPXTG penta-peptide motif and forms a new peptide bond betweenthe N-terminal glycine of the G_(n) stretch (n>2) and the T residue. TheC-terminal G residue of the LPXTG motif (here highlighted in boldfaceprint) is removed in the transpeptidation reaction. (b) Conversely, inorder to achieve C-terminal conjugation of a payload to a protein, whichis the preferred method for conjugation of payloads, particularlytoxins, to antibodies (see FIG. 6), the LPXTG sortase recognitionpenta-peptide motif needs to be added to the C-terminal end of theimmunoligand/binding protein (e.g. by recombinant protein expressiontechnology, as described in the Examples), and the payload needs to bemodified with a short glycine stretch (G_(n), with n>2, typically 3-5glycines). As described under (a), addition of sortase A fromStaphylococcus aureus will then catalyze the transpeptidation of theGn-stretch to the LPXTG motif, whereby the terminal G residue of theLPXTG motif (in boldface) will be removed.

FIG. 2: This figure illustrates the principle of intein (a) andsplit-intein (b) mediated transpeptidation. (a) Inteins can occur asso-called “protein-introns” in precursor proteins, where they separateN-terminal and C-terminal parts of a mature protein, which are generallycalled N-extein and C-extein. The intein “protein-intron” can catalyzethe breakage of the peptide bond between the intein and the C-extein andthe formation of a new peptide bond between the N-extein and C-extein bytransfering the N-terminal amino acid of the C-extein to the C-terminalamino acid of the N-extein in a transpeptidation reaction. The result ofthe reaction is the removal of the intein “protein-intron” from theprecursor protein and the generation of a mature protein with a newlycreated peptide bond between the N-extein and C-intein domains. (b) Theintein activity has also been described to be separable into distinctdomains, that can be attached to different proteins, for which thisintein variation has been termed split-intein. The N-int and C-intdomains of the split intein form a non-covalent structural complex, thatcan perform the same transpeptidation reaction as a contiguous intein,on the attached N-extein and C-extein domains that are then in spatialproximity and part of the complex. The result of the transpeptidation ofN-int and C-int split-intein reaction is then a “proteintrans-splicing”, or essentially a protein ligation between the N-exteinand C-extein domains, by formation of a novel peptide bond.

FIG. 3: This figure illustrates how particular split inteins that arecharacterized by either an extremely short C-int domain or an extremelyshort N-int domain can be used to conjugate any payload to animmunoligand (or binding protein), including small molecular entities,because short amino acid stretches can be synthesisze chemically and caneasily be attached to small molecular entities by conventional chemicalcoupling. (a) This part of the illustration shows the use of the SspGyrB S11 split intein (described in Appleby et al. (2009)) for theC-terminal conjugation of a payload to an immunoligand/binding protein.Here the C-int domain is only 6 amino acids long and comprises the aminoacid sequence GVFVHN, as indicated. However, as there need to be somepeptides that are the equivalent of an C-extein domain, additional aminoacids need to be added, of which the first one needs to be a serine orcysteine amino acid residue, whereas the remaining amino acids can bechosen. This is indicated by the SX_(n) symbol, which means that a shortamino acid stretch lead at the N-terminal side by serine and followed byn amino acids (n>2, preferably 5), which can be any of the 20 naturallyoccuring amino acids (therefore indicated as X). Thus, as described inthe Example, a short 12 amino acid stretch comprising a 6 amino acidmini C-int domain and 6 amino acid C-ext amino acid stretch aresufficient to allow the N-int/C-int complex to catalyze thetranspeptidation from the asparagine-serine peptide bond in theGVFVHN-SX_(n) (X any amino acid, n>2, preferably 5) to the peptide bondbetween the N-extein and N-int transition. This will result in aC-terminally conjugated immunologand/binding protein with the payloadattached via the short C-extein amino acid stretch. (b) This part of theillustration shoas the use of the Ssp DnaX split intein (described inSong et al. (2012)), which can be separate into a very short, 11 aminoacid N-int domain and a 139 aa C-int domain for N-terminal conjugationof a payload to an immunologand/binding protein. As indicated here, thisonly requires the synthesis and coupling of a short 11 amino acid N-intdomain to any payload (or the addition by recombinant proteintechnology), which then allows the specific conjugation of the payloadto the N-terminus of any immunoligand or protein, that has a 139 aminoacid long Ssp DnaX C-int domain fused to the N-terminus. The result ofthis reaction is then a N-terminally conjugated immunoligand/bindingprotein. Therefore, like in the case of sortase transpeptidation, wherethe N- or C-terminal conjugation only depends on the arrangement of theLPXTG and G_(n) peptide motifs with regard to protein and payload, splitinteins can also mediate site-specific N- and C-terminal conjugation ofproteins with short peptide modified payloads, and by exploit short miniC-int, or mini N-int peptide domains, like those of Ssp GyrB and SspDnaX split inteins, respectively.

FIG. 4: (a) This Figure illustrates the utility of adding additionalaffinity purification and/or detection tags in addition to a sortase tagin the conjugation of payloads to immunoligands. (a) this part of theFigure shows how an additionally added amino acids representing a 6×Hispurification tag (HHHHHH), a Myc-detection tag (EQKLISEEDL) and astrepII affinity purification tag (WSHPQFEK), as described in theExamples are removed in the course of the C-terminal payload conjugationvia Staphylococcus aureus sortase A transpeptidase. This allows toselect for the conjugated product, if Ni-NTA affinity resins (for the6×His-tag) or streptactin afifnity resins (for the strep II-tag) areemployed to seperate non-conjugated substrate from conjugated product.This combination of tags is only provided by way of Example.

(b) This Figure illustrates that the use of affinity purification tagsis particularly useful to select/purifiy completely conjugated productin the case of multimeric proteins, like antibodies as illustrated here.As also provided in the examples, antibodies can be modified withspecific conjugations sites at heavy and light chains, and if themodification is targeted to the C-termini of IgH and IgL chains, then upto four payloads may be conjugated to the antibody. The addition of (a)further affinity purification tag(s), e.g. as described in FIG. 4(a)allows to bind incompletely conjugated product, that may only have one,two, or three (as illustrated here) payloads conjugated to the antibody,still bind to the respective affinity purification resin, and can thuseasily be separated from the fully payload-conjugated product. Thisparadigm is of course also applicable to intein-modified immunologands,and not only to sortase-motif-modified immunologands, as depicted here.

FIG. 5: This figure illustrates a variation of the sortase-mediatedconjugation that can also be applied, in which the sortase-enzyme is notadded as a separate recombinant protein to the sortase taggedimmunologand and glycine-stretch modified payload, but where theenzymatic sortase domain is expressed as a fusion protein C-terminal tothe LPXTG sortase tag. The sortase enzyme domain will be inactive aslong as it is not incubated with glycine-stretch modified payload (orsubstrate). As soon as glycine-stretch modified substrate (or herepayload) is added to such a construct, the fused sortase domain willcatalyze the transpeptidation of glycine-payload substrate to the LPXTGsortase tag, by cleaving the protein between the threonine-4 andglycine-5 position of the LPXTG tag, and thereby removing the sortaseenzyme domain with additional affinity purification tags, that can beadded optionally, as depicted here. This procedure has the advantagethat, similar to the addition of catalytically active split-inteindomains, the sortase enzyme domain can be expressed by recombinantprotein technology as an integral component of the immunoligand to beconjugated.

FIG. 6: (a) This figure illustrates the use of different transpeptidases(here sortase and split-intein), in order to simultaneously conjugatedifferent payloads to different subunits of a multimeric protein, likee.g., as depicted here, the heavy and the light chains of an antibody.In this selected example, the C-termini of the heavy chains are modifiedwith the N-int domain of Ssp GyrB (as provided in Example 2), while thelight chains are modified with the sortase A penta-peptide motif LPXTG(as provided in Example 1, the additional tags are omitted forsimplicity). Incubation with a glycine-stretch modified payload A andwith a C-int-domain modified payload B and sortase enzyme will allow thesimultaneous and selective conjugation of payload B to the heavy chainsand payload A to the light chains. If payloads A and B are toxinsaddressing different cellular pathways, this strategy could generatemore potent anti-cancer drugs, as conventional ADCs, only containing asingle toxin moiety. (b) This figure illustrates the use of differentsortase enzymes (here sortase A and sortase B from Staphylococcusaureus), in order to simultaneously conjugate different payloads todifferent subunits of a multimeric protein, like e.g., as depicted here,the heavy and the light chains of an antibody. In this selected example,the C-termini of the heavy chains are modified with the pentapeptiderecognition motif for sortase B, NPQTN, while the light chains aremodified with the sortase A penta-peptide motif LPXTG. Sequentialconjugation of glycine-stretch modified payloads A and B with sortase Aand sortase B will allow the simultaneous and selective conjugation ofpayload B to the heavy chains and payload A to the light chains(remaining peptide sequences from LPXTG and NPQTN are omitted in theconjugated structure for simplicity). If payloads A and B are toxinsaddressing different cellular pathways, this strategy could generatemore potent anti-cancer drugs, as conventional ADCs, only containing asingle toxin moiety.

FIG. 7: SDS-PAGE (a.) and Western-blot (b.) analysis of recombinantenzymatically active sortase A fragment of Staphylococcus aureus. (a.)Lane 1 in the SDS-PAGE contains BSA (ca. 66.4 kD), Lane M₁ containsprotein molecular weight standard of Genscript (Cat.-Nr.: MO0505), Lane2 contains His-tag purified recombinant sortase A fragment ofStaphylococcus aureus. The proteins in the SDS-PAGE are stained withCommassie blue. (b.) The Western-blot was developed with an anti-Hisantibody (Genscript Cat.-Nr.: AO0186). Lane 3 contains His-tag purifiedrecombinant sortase A fragment of Staphylococcus aureus. Lane M₂contains molecular weight standard of Genscript (Cat.-Nr.: MM0908).

FIG. 8: Hydrophobic Interaction Chromatography (HIC) analysis ofDM1-toxin conjugated Tras-HC-GS-LHS (A) and Tras-LC-GS-LHS (B). DAR1indicates drug to antibody ratio of 1; DAR2 indicates a drug to antibodyratio of 2.

FIG. 9: Dose response of cytotoxic effects of the indicated ADCs onHER2-overexpressing SKBR3 (A) and HER2-negative T47D-5R cells (B). Cellswere incubated with serial dilutions of ADCs for 3 days, after whichcell viability was detected by CellTiter-Glo® Luminescent Solution(Promega). LC: DM1-sortaseA-conjugated Tras-LC-GS-LHS; HC:DM1-sortaseA-conjugated Tras-HC-GS-LHS.

FIG. 10: Sortase A-mediated conjugation of Gly₅-FITC to mAb Ac10variants with or without GS peptide spacer. Serial dilutions of SortaseA were used to conjugate Gly₅-FITC to mAb Ac10-HC-GS-LHS/LC-GS-LHS (A)and mAb Ac10-HC-LS/LC-LS (B) under otherwise identical conditions.Reaction products were seperated by size on denaturing, reducingSDS-PAGE gels. FITC was visualized by placing the gels on a UV box.Sortase A concentrations used were: lanes 1, 9: 50 μM; lanes 2, 10: 25μM; lanes 3, 11: 12.5 μM; lanes 4, 12: 6.25 μM; lanes 5, 13: 3.13 μM;lanes 6, 14: 1.56 μM; lanes 7, 15: 0.78 μM; lanes 8, 16: 0.39 μM.

FIG. 11: Influence of peptide spacer length on light chain conjugationefficiency. Serial dilutions of Sortase A were used to conjugateGly₅-FITC to mAbs Ac10-HC-LS/LC-GS-LS (A, left), Ac10-HC-LS/LC-GGS-LS(A, right), Ac10-HC-LS/LC-GGGS-LS (B, left) and Ac10-HC-LS/LC-GGGGS-LS(B, right) under otherwise identical conditions. Reaction products wereseperated by size on denaturing, reducing SDS-PAGE gels. FITC wasvisualized by placing the gels on a UV box. Sortase A concentrationsused were: lanes 1, 8, 15, 22: 25 μM; lanes 2, 9, 16, 23: 12.5 μM; lanes3, 10, 17, 24: 6.25 μM; lanes 4, 11, 18, 25: 3.13 μM; lanes 5, 12, 19,26: 1.56 μM; lanes 6, 13, 20, 27: 0.78 μM; lanes 7, 14, 21, 28: 0.39 μM

FIG. 12: Analysis of sortaseA vc-PAB-MMAE toxin heavy-chain-conjugatedADC of mAb Ac10 by hydrophobicity interaction chromatography (HIC),which is able to differentiate unreacted substrate (DAR0=0 drug toantibody ratio), substrate in which one of the two heavy chains has beenconjugated (DAR1=1 drug to antibody ratio), and substrate in which bothmodified heavy chains have been conjugated (DAR2=2 drugs to antibodyratio), as indicated. Panel A shows the HIC profile after a standardsortase A mediated conjugation of HC modified Ac10 mAb, in which stillDAR0 and DAR1 species are detectable, next to the desired DAR2 product.Panel B shows the HIC profile after 4 passes of the ADC preparationanalyzed in Panel A over a StrepTactin® affinity purification column.

FIG. 13: Analysis of synthesized Gly_(s)-modified alpha-amanitin toxin(a.) and Gly_(s)-modified maytansin toxin (b.). In each of the panelsa.) and b.) the snthesized structure is provided on top, with the fiveglycines highlighted by a box. The analysis of each compound by massspectrometry and reverse-phase HPLC is provided below. a.) The expectedmass of the Gly₅-modified alpha-amanitin toxin is 1302.07 D, theobserved mass is 1325.38 D, corresponding to Ms+Na⁺. The RP-HPLC profileindicates a purity of >95%. b.) The expected mass of the Gly₅-modifiedmaytansine toxin is 991.41 D, the observed mass is 957.69 D,corresponding to Ms +Nat The RP-HPLC profile indicates a purity of >95%.

FIG. 14: Structures of 5×Glycine (Gly₅) modified toxins that either havebeen synthesized by Concortis, San Diego, Calif., U.S. (structures 1-6,and 9), or that can be synthesized (structures 7 & 8), demonstratingthat any toxin can be functionalized for sortase mediated enzymaticconjugation, if either 5 glycines are attached to the toxins (as shownhere), or any number of glycine residues greater or equal than oneglycine. Glycine-modified toxins can either be synthesized containingadditional validated linker/spacer structures as provided in structures1-3 in FIG. 14 a.), potentially adding certain additional functionality(e.g. cleavability in certain subcellular compartments) or withoutadditional linkers, as depicted in structures 4-6 in FIG. 14 b.). Ifseveral reactive groups are available at a given toxin, like e.g. in thecase of alpha-amanitin toxin, glycine residues can be added to thesedifferent groups as exemplified in structures 7-9 in FIG. 14c .).

FIG. 15: Tumor volumes as determined in Example 12. The resultsdemonstrate that sortase conjugated ADCs, using identical antibody andtoxin moiety, have comparable tumor killing activity in comparison tocommercially available chemically conjugated Kadcyla®.

FIG. 16: Gels stained with Coomassie blue as described in example 13 Thedata shows the unexpected finding that Sortase A-mediated conjugation ofantibodies in crude cell culture supernatant was as efficient as that ofpurified antibody.

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1-24. (canceled)
 25. An immunoligand/payload conjugate produced by amethod comprising a) providing an immunoligand selected from the groupconsisting of: an antibody, (i) an antibody-based binding protein beinga protein containing at least one antibody-derived V_(H), V_(L), orC_(H) immunoglobulin domain, (ii) an antibody fragment binding to areceptor, antigen, growth factor, cytokine and/or hormone, and/or (iii)an antibody mimetic selected from the group consisting of DARPins,C-type lectins, A-domain proteins of S. aureus, transferrins,lipocalins, 10th type III domains of fibronectin, Kunitz domain proteaseinhibitors, affilins, gamma crystallin derived binders, cysteine knotsor knottins, thioredoxin A scaffold based binders, nucleic acidaptamers, artificial antibodies produced by molecular imprinting ofpolymers, and stradobodies, and, b) enzymatically conjugating at leastone payload to the immunoligand by a sequence-specific sortase or acatalytic domain thereof, wherein the payload is a toxin having amolecular weight not exceeding 2,500 Dalton, wherein either theimmunoligand comprises a sortase recognition motif and the toxin ismodified with a Gly_(n)-modification, wherein n >1, or the toxincomprises a sortase recognition motif and the immunoligand is modifiedwith a Gly_(n)-modification, wherein n >1.
 26. The immunoligand/payloadconjugate of claim 25, wherein the immunoligand binds at least oneentity selected from the group consisting of a receptor, an antigen, agrowth factor, a cytokine, and a hormone.
 27. The immunoligand/payloadconjugate of claim 25, wherein the immunoligand is conjugated to atleast two different payloads, at least one of which is the toxin, andwherein said immunoligand has at least two subunits each beingconjugated to a payload.
 28. The immunoligand/payload conjugate of claim25, wherein the immunoligand comprises two subunits, at least one ofwhich has a peptide spacer appended to the C-terminus thereof, saidspacer comprises at least two amino acids.
 29. The immunoligand/payloadconjugate of claim 28, wherein the spacer is 2-5 amino acids.
 30. Theimmunoligand/payload conjugate of claim 25, wherein the ratio ofimmunoligand and payload is stoichiometrically defined.
 31. Theimmunoligand/payload conjugate of claim 25, wherein the conjugation ofthe payload to the immunoligand is site-specific.
 32. Theimmunoligand/payload conjugate of claim 25, wherein the sortaserecognition motif is LPXTG (SEQ ID NO:27) or NPQTN (SEQ ID NO:28). 33.An immunoligand/payload conjugate comprising a) an immunoligand selectedfrom the group consisting of: (i) an antibody, (ii) an antibody-basedbinding protein being a protein containing at least one antibody-derivedV_(H), V_(L), or C_(H) immunoglobulin domain, (iii) an antibody fragmentbinding to a receptor, antigen, growth factor, cytokine and/or hormone,and/or (iv) an antibody mimetic selected from the group consisting ofDARPins, C-type lectins, A-domain proteins of S. aureus, transferrins,lipocalins, 10th type III domains of fibronectin, Kunitz domain proteaseinhibitors, affilins, gamma crystallin derived binders, cysteine knotsor knottins, thioredoxin A scaffold based binders, nucleic acidaptamers, artificial antibodies produced by molecular imprinting ofpolymers, and stradobodies, and, b) at least one a payload being a toxinhaving a molecular weight not exceeding 2,500 Dalton, wherein theconjugate further comprises at least one linker between the payload andthe immunoligand, said linker comprising a peptide motif that is asortase recognition motif or a derivative thereof, and said linker beingconjugated to the C-terminus of at least one peptide chain of theimmunoligand.
 34. The immunoligand/payload conjugate according to claim33, wherein in the derivative of the sortase recognition motif, theC-terminal amino acid residue of the original sortase recognition motifis replaced by a G residue.
 35. The immunoligand/payload conjugateaccording to claim 34, wherein the derivative of the sortase recognitionmotif is -LPXTG_(n)- or -NPQTG_(n)-.
 36. The immunoligand/payloadconjugate of claim 33, wherein the immunoligand binds at least oneentity selected from the group consisting of a receptor, an antigen, agrowth factor, a cytokine, and a hormone.
 37. The immunoligand/payloadconjugate of claim 33, wherein the immunoligand is conjugated to atleast two different payloads, at least one of which is the toxin, andwherein said immunoligand has at least two subunits each beingconjugated to a payload.
 38. The immunoligand/payload conjugate of claim33, wherein the immunoligand comprises two subunits, at least one ofwhich has a peptide spacer appended to the C-terminus thereof, saidspacer comprises at least two amino acids.
 39. The immunoligand/payloadconjugate of claim 38, wherein the spacer is 2-5 amino acids.
 40. Theimmunoligand/payload conjugate of claim 33, wherein the ratio ofimmunoligand and payload is stoichiometrically defined.
 41. Theimmunoligand/payload conjugate of claim 33, wherein the conjugation ofthe payload to the immunoligand is site-specific.
 42. A lowmolecular-weight (LMW) payload, wherein the payload is a toxin having amolecular weight not exceeding 2,500 Dalton, and the toxin is modifiedwith a Gly_(n)-modification, wherein n>1.
 43. The LMW payload of claim42, wherein the payload is selected from Maytansine, MonomethylAuristatin and Alpha-amanitin or functional derivatives thereof.
 44. Amethod of treating a human or animal subject suffering from or being atrisk of developing a pathologic condition, said method comprisingadministering to said subject the immunoligand/payload conjugate ofclaim
 25. 45. The method of claim 44, wherein the pathologic conditionis selected from the group consisting of neoplastic disease, autoimmunedisease, neurodegenerative disease, and infectious disease.
 46. A methodof treating a human or animal subject suffering from or being at risk ofdeveloping a pathologic condition, said method comprising administeringto said subject the immunoligand/payload conjugate of claim
 33. 47. Themethod of claim 46, wherein the pathologic condition is selected fromthe group consisting of neoplastic disease, autoimmune disease,neurodegenerative disease, and infectious disease.